Bull. Southern California Acad. Sci.
122(1), 2023, pp. 1-18
© Southern California Academy of Sciences, 2023
Urchin Gonad Response to Kelp Forest Restoration on the
Palos Verdes Peninsula, California
Benjamin C. Grime,!:** Rilee Sanders,!-? Tom Ford,! Heather Burdick,! and
Jeremy T. Claisse”-4
The Bay Foundation, Los Angeles, CA 90293, US'A
* Department of Biological Sciences, California State Polytechnic University, Pomona,
CA 91768, USA
Scripps Institution of Oceanography, La Jolla, CA 92093, USA
4Vantuna Research Group, Occidental College, Los Angeles, CA 90041, US'A
Abstract.—Along the Palos Verdes Peninsula in southern California, high densities of
Strongylocentrotus purpuratus (purple sea urchin) have consumed almost all macroal-
gae on large expanses (61 ha) of rocky reef habitat, creating “urchin barrens.”
Mesocentrotus franciscanus (red sea urchin) harvesting comprises an important
fishery in the region, as their gonads are sold as a high-value sushi product called
“uni.” However, with a lack of macroalgal food resources, urchins in barrens
are smaller and exist in a starved state, meaning little, if any, gonad product is
available to the fishery. To restore local kelp forests and increase gonad biomass
available to the M. franciscanus fishery, beginning in October 2013, S. purpura-
tus were culled in barrens to a target density of 2 per m? across 5.2 ha of rocky
reef on the Palos Verdes Peninsula. Mesocentrotus franciscanus were collected
from urchin barren, restoration, and kelp reference sites from April to Novem-
ber 2014 to compare differences in gonad production among the three site types.
Culling S. purpuratus resulted in the recovery of normal seasonal M. francis-
canus gonad production throughout the 8-month study. Mesocentrotus franciscanus
gonad weights at a given test diameter length in restoration sites were equivalent to,
and sometimes exceeded, the gonad production of those from the kelp reference sites.
The urchin test length distributions of collected M. franciscanus were consistently
smaller at urchin barren sites than at kelp reference sites, while those in restoration
sites generally fell in between.
Giant kelp (Macrocystis pyrifera) forests are among the most productive and diverse
ecosystems in the world (Dayton 1985; Graham 2004). Kelps are autogenic engineers, pro-
viding physical structure for a high diversity of flora and fauna. In addition, they are a food
source for a wide variety of taxa, contributing to the food web through direct grazing and
as dissolved organic materials (Graham 2004; Duarte et al. 2022). Yet, the combined effects
of overfishing, pollution, and increasing frequency of warm water events have led to the
destructive grazing of kelp by sea urchins and the formation of “urchin barrens” that can
last decades (Steneck et al. 2002; Cavanaugh et al. 2019; Rogers-Bennett and Catton 2019;
Kawamata and Taino 2021) (Fig. 1). These barren reefs are largely devoid of macroal-
gae and covered instead by high proportions of bare rock and encrusting coralline algae
* Corresponding author: grime.benjamin@gmail.com
1
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2 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Fig. 1. (A) High densities of S. purpuratus in an urchin barren state, (B) kelp forest state in southern
California, (C) partially dissected Mesocentrotus franciscanus from a kelp forest site, and (D) extracted M.
franciscanus urchin gonad (i.e., uni).
(Dayton 1985; Steneck et al. 2002; Cavanaugh et al. 2011; Rogers-Bennett and Catton
2019; Gizzi et al. 2021; Williams et al. 2021).
Feedback mechanisms on temperate rocky reefs increase the resilience of both healthy
kelp forest and urchin barren states (Levin and Lubchenco 2008; Baskett and Salomon
2010; Filbee-Dexter and Scheibling 2014). The kelp forest state is maintained by positive
feedback mechanisms that prevent high densities of urchins from forming and destructively
grazing (Baskett and Salomon 2010; Filbee-Dexter and Scheibling 2014). In the presence
of abundant predators, urchins exhibit cryptic behavior and lower densities are maintained
(Nichols et al. 2015). However, overfishing of urchin predators can reduce top-down con-
trol of urchin abundance, leading to a phase shift where sea urchins have a greater impact
on the ecosystem dynamics (Jackson et al. 2001; Steneck et al. 2002; Filbee-Dexter and
Scheibling 2014; Melis et al. 2019). When urchin densities subsequently increase above a
critical threshold, the transition from a kelp forest to an urchin barren occurs rapidly, re-
sulting in destructive grazing and prevention of substratum growth (Baskett and Salomon
2010; Filbee-Dexter and Scheibling 2014; Karatayev and Baskett 2020; Kawamata and
Taino 2021). Evidence from various studies show a phase shift threshold from an algal
dominated system to barren formation occurs when urchin biomass exceeds 700 g/m’,
though exact numbers are highly dependent on region specific dynamics (Ling et al. 2015).
An urchin barren state is then maintained by positive feedback mechanisms that promote
sea urchin recruitment, settlement, and overgrazing, all of which inhibit kelp settlement
(Filbee-Dexter and Scheibling 2014; Sangil and Hernandez 2022).
vz0z Jaquieoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-pESr-Z9LZVLOZ86LE/L/L/2z Lpd-sjomesunslinqseos/woo sseidualje uelpuew//:dyy Woy pepeojumoq
URCHIN GONAD RESPONSE TO RESTORATION 3
Destructive sea urchin grazing is the leading cause of kelp deforestation in the world
(Steneck et al. 2002) and has multi-trophic level impacts on species’ use of kelp forest
habitat resources (Graham 2004; Rogers-Bennett and Catton 2019). Urchin barrens occur
globally in most regions where kelp forests exist (Steneck et al. 2002; Gagnon et al. 2004;
Ling et al. 2015). In southern California, urchin barrens have been present on the Palos
Verdes Peninsula since the 1950s (North 1963; Foster and Schiel 2010). Surveys conducted
in the late 1960s had described a near total absence of adult giant kelp on the Palos Verdes
Peninsula (Wilson et al. 1977; Foster and Schiel 2010) likely due to the combined influence
of increased coastal development, sedimentation, urban runoff, pollution, and direct kelp
removal from storms (North 1963; Dayton 1985; Steneck et al. 2002; Ford and Meux 2010;
Foster and Schiel 2010). Following this large-scale kelp reduction, a transition to an urchin
dominated system further prevented kelp recruitment (North 1963; Foster and Schiel 2010;
Filbee-Dexter and Scheibling 2014). As of 2012, there were 61 ha of rocky reef persisting
in an urchin barren state on the Palos Verdes Peninsula (Claisse et al. 2013).
Kelp forest loss and the resulting urchin barrens have systemic ecological implications,
as well as significant economic impacts on sea urchin fisheries (Claisse et al. 2013; Rogers-
Bennett and Catton 2019). Historically, the Mesocentrotus franciscanus (red sea urchin)
fishery was consistently one of the largest fisheries in California by annual tonnage har-
vested (NMFS 2018). Mesocentrotus franciscanus are harvested for their gonads, as a high
value sushi product called “uni” (Rogers-Bennett 2007; Teck et al. 2018). In urchin bar-
rens, particularly when coexisting with high densities of S. purpuratus, M. franciscanus
gonads are substantially underdeveloped (Harrold and Reed 1985; Kato and Schroeter
1985; Rogers-Bennett et al. 1995; Spindel et al. 2021), resulting in a decreased amount of
gonad product available to the fishery (Claisse et al. 2013). For example, in northern Cal-
ifornia, the M. franciscanus fishery remained stable from 2000-2014, but recent extensive
losses of kelp, combined with increases in S. purpuratus dominated urchin barrens, resulted
in urchin gonad biomass declines that eventually led to the collapse of the M. franciscanus
commercial fishery (Rogers-Bennett and Catton 2019; Angwin et al. 2022).
In an urchin barren state, urchin gonad biomass is reduced due to the lack of macroalgal
food available (Bernard 1977; Rogers-Bennett 2007). Other urchin health metrics, includ-
ing growth rates, test diameter size, density, and biomass, can also be negatively affected by
reductions in food resources (Ebert 1967; Claisse et al. 2013; Teck et al. 2017). In addition
to using their gonads for reproduction, urchins also use gonads to store nutrients as fats
and carbohydrates within the tissue (Doezema and Phillips 1970), having the ability to re-
sorb their gut and gonad complex for energy (Giese et al. 1966; Pearse et al. 1970; Kato and
Schroeter 1985; Rogers-Bennett et al. 1995). With the almost complete absence of macroal-
gal food availability in barrens, urchins exist in a starved state (Kato and Schroeter 1985)
through metabolic depression, which induces morphological changes (Smith and Garcia
2021; Spindel et al. 2021), permitting the urchins to survive by feeding on plankton and
diatoms (Pearse et al. 1970; Kato and Schroeter 1985; Hernandez et al. 2011). However,
most populations of urchins in barrens have poor health, reduced gonads, and smaller test
diameter sizes (Pearse et al. 1970; Ling and Johnson 2009; Claisse et al. 2013; Williams
et al. 2021).
In kelp forests, urchin gonad development and spawning follow a seasonal pattern (Ebert
et al. 1994; Hernandez et al. 2011; Teck et al. 2018), although there is high variation even
within the same species over relatively small spatial scales (Kato and Schroeter 1985). In
California, increased seasonal gonad production typically occurs in late fall or early winter
seasons as a direct result of higher drift kelp availability due to natural kelp forest growth
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4 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
and kelp recovery following winter and spring storms (Cavanaugh et al. 2011; Teck et al.
2018). Spawning typically occurs directly following a period of peak kelp abundance and
gonad production when sea urchins are investing energy into developing gonads for re-
production (Ebert et al. 1994; Teck et al. 2018). While an increase in gonad production is
an indicator or cue to spawn, there is evidence that gonad development also occurs inde-
pendently for energy storage, suggesting there are other seasonal cues at play that induce
spawning (Hernandez et al. 2011). It is therefore necessary to consider both direct food
availability and other seasonal influences on gonad production when considering manage-
ment efforts to maintain both the commercial urchin fishery and the kelp forest state (Teck
et al. 2017; 2018), as well as to inform restoration efforts (Claisse et al. 2013).
A variety of restoration techniques have been implemented to try to restore barrens to
kelp forests, and most have involved removing the main driver (i.e., sea urchins) of kelp
deforestation (Flukes et al. 2012; Eger et al. 2020; 2022; Layton et al. 2020). On the Palos
Verdes Peninsula, kelp forest restoration efforts aimed at reducing barren-forming urchin
densities have been ongoing since the early 2000s (Ford and Meux 2010; Williams et al.
2021). In this area, the vast majority of urchins in these barrens are S. purpuratus (Claisse
et al. 2013). Beginning in 2013, commercial urchin harvesters were employed as part of a
large-scale kelp restoration effort along the Palos Verdes Peninsula to reduce S. purpuratus
density to 2 per m? through urchin culling. The commercially important M. franciscanus
were not culled in an effort to increase production for the local commercial urchin fishery
(Claisse et al. 2013).
The present study examines how M. franciscanus gonad biomass production responded
to the effects of culling S. purpuratus in barrens to restore kelp forests along the Palos
Verdes Peninsula in 2014, prior to a natural urchin mass mortality event at the end of
that year (Williams et al. 2021). We compare changes in M. franciscanus gonad biomass
over time collected from three site types: kelp forest reference sites, urchin barren sites,
and restoration sites following urchin density reduction from active culling. Sea urchin
nutrition and gonad production is positively influenced by increases in kelp availability
(Pearse et al. 1970; Claisse et al. 2013; Teck et al. 2018). Further, kelp forests have the
ability to rebound rapidly following disturbances (Cavanaugh et al. 2011; Williams et al.
2021), suggesting gonad production should also increase rapidly following restoration
activities.
Materials and Methods
All project activities occurred on the Palos Verdes Peninsula, located in Los Angeles
County, California (Fig. 2). Habitat areas within sites were initially classified as urchin
barrens or kelp forests by a previous study (Claisse et al. 2013). The three site types in this
study were designated as urchin barren, kelp reference, and restoration. Urchin barrens
were identified as areas of rocky reef almost entirely devoid of macroalgae, characterized
by high percent cover of bare substrate, encrusting coralline algae, and high densities of S.
purpuratus (Claisse et al. 2013; Gizzi et al. 2021). Restoration activities occurred at Hon-
eymoon Cove from 29 October 2013, to 7 April 2014, and at Underwater Arch from 10
September 2013, to 5 August 2014 (Table 1). Divers used rock hammers to reduce S. pur-
puratus densities in barrens to a target density of < 2/m?. Divers left M. franciscanus in
place as they were not overabundant and are economically valuable to the urchin fishery
(Claisse et al. 2013). Given the large spatial scale of this restoration effort in the region
(22.8 ha total target area), and the nature of the labor force (e.g., three commercial urchin
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URCHIN GONAD RESPONSE TO RESTORATION )
LOS ANGELES
COUNTY
33.79°N
Palos Verdes
Rocky Point Pe n i nsu la
Lunada Bay
Honeymoon Cove
33.76°N
Marguerite Cove
Underwater Arch Cove
Site Type
ei Barren
& Kelp
33.73°N - @ Restoration
118.43°W 118.39°W 118.35°W
Fig. 2. Urchin survey and collection sites on the Palos Verdes Peninsula, California. Site types are
designated by color. Some areas within the restoration sites were used as urchin barren collection sites prior
to restoration activities beginning in those areas (Sub-site IDs in Table 1).
diver teams, The Bay Foundation non-profit employees, volunteers), restoration of sites
was initially intended to occur sequentially. However, practical considerations resulted in
restoration efforts occurring concurrently at some reefs, and divers would often return to
sites after a period of weeks to months to monitor and cull additional S. purpuratus urchins
found in small high-density patches until the entire site was considered ‘restored’ with a tar-
get density of < 2/m? (restoration end dates listed in Table 1). It is also important to note
that S. purpuratus densities within most of the restoration areas were typically reduced to
the target density months prior to the restoration end date.
In 2013, prior to the start of restoration activities at a given area (Sub-site ID,
Table 1), pre-restoration monitoring was conducted at urchin barren sites to be restored per
California Department of Fish and Wildlife (CDFW) standards in accordance with the
terms of the Scientific Collecting Permit issued to the Bay Foundation (TBF; S-183390001-
19133-001). Urchin barren sites were divided into 30 m x 30 m blocks, each comprised of
15 parallel and adjacent (30 m x 2 m) transects. TBF biologists counted S. purpuratus along
five of the fifteen 30 m x 2 m transects per block to estimate pre-restoration density of S.
purpuratus of the block (range 57.2 — 21.2/m/?; Table 1).
Post-restoration monitoring was conducted once the team doing the restoration reported
to TBF that S. purpuratus densities within the block had been reduced to the target density
of < 2/m?. All 15 transects within each 30 m x 30 m block were then surveyed by TBF staff
to ensure that no pockets of high-density S. purpuratus remained at the site, and if this was
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6 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Table 1. Mesocentrotus franciscanus collection and restoration activities (1.e., culling S. purpuratus).
Sub-site ID and associated latitude and longitude give the specific areas for each collection within the overall
site location (Site Name): Honeymoon Cove (HMC), Lunada Bay (LB), Underwater Arch Cove (UAC),
Marguerite Cove (MC), Rocky Point (RP). Restoration Start is the first day restoration actions began, and
Restoration End is the last day restoration actions occurred in a specific Sub-site ID. Pre and Post refers to
the S. purpuratus density before and after culling in a specific Sub-site ID. n is the number of M. franciscanus
collected from each location on each collection date.
Site Site Restoration Pre Restoration Post
Collection Date Type Name Latitude Longitude Sub-site ID Start (No./m7) End (No./m?) n
29 Apr 2014 Barren HMC 33.7648 -118.4232 HMC_B1_B - - - - 47
Kelp LB 33.7690 -118.4255 LB_K1_K - - - - 30
Rest. HMC 33.7637 -118.4234 HMC_T1_R = 29 Oct 2013 54.4 26 Feb 2014 16 49
Rest. UAC = 33.7521 -118.4157 UAC_J1_R = 25 Oct 2013 48.9 11 Apr 2014 2.3 48
28 May 2014 Barren UAC = 33.7539 -118.4156 UAC_B2_B - - - - 26
Kelp LB 33.7691 -118.4246 LB_K2 K - - - - 37
Rest. HMC 33.7637 -118.4234 HMC_T1_R = 29 Oct 2013 54.4 26 Feb 2014 1.6 52
Rest. HMC 33.7648 -118.4247 HMC_R1R_ 01 Nov 2013 42.6 21 Mar 2014 1.6 51
Rest. UAC = 33.7521 -118.4157 UAC_JI_R = 25 Oct 2013 48.9 11 Apr 2014 2.3 50
Rest. UAC 33.7541 -118.4163 UAC_3_R 10 Sep 2013 57.211 Dec 2013 3.1 62
26 June 2014 Barren UAC = 33.7536 -118.4150 UAC_B3_B - - - - 36
Kelp LB 33.7664 -118.4257 LB_K3_K - - - - 33
Rest. HMC 33.7637 -118.4234 HMC_T1_R = 29 Oct 2013 54.4 26 Feb 2014 1.6 38
Rest. UAC = 33.7521 -118.4157 UAC_J1_R = 25 Oct 2013 48.9 11 Apr 2014 2.3 39
22 July 2014 Barren UAC = 33.7525 -118.4148 UAC_B4_ B - - - - 29
Kelp LB 33.7680 -118.4256 LB_K4 K - - - - 50
Rest. HMC 33.7650 -118.4250 HMC_R1R_ 01 Nov 2013 42.6 21 Mar 2014 16 43
Rest. UAC = 33.7521 -118.4157 UAC_J1_R = 25 Oct 2013 48.9 11 Apr 2014 2.3 53
30 Oct 2014 Barren MC 33.7556 -118.4174 MC_B5_B - - - - 58
Kelp RP 33.7714 -118.4284 RP_K5_K - - - - 42
Rest. HMC 33.7643 -118.4234 HMC_T2_R_ 11 Mar 2014 21.2 07 Apr 2014 1.9 47
Rest. UAC = 33.7539 -118.4158 UAC_WI_R 11 Dec 2013 34.6 05 Aug 2014 1.5 60
18 Nov 2014 Barren MC 33.7560 -118.4166 MC_B6_B - - - - 55
Kelp LB 33.7662 -118.4254 LB _K6_K - - - - 54
Rest. HMC 33.7640 -118.4234 HMC_T1_R_ 29 Oct 2013 54.4 26 Feb 2014 1.6 55
Rest. UAC = 33.7522 -118.4154 UAC_J1_R — 25 Oct 2013 48.9 11 Apr 2014 2.3 54
the case, then that was recorded as the restoration end date. Post-restoration S. purpuratus
densities ranged from 3.1 to 1.5/m? across blocks (Table 1).
To examine differences in gonad production between site types over time, TBF staff and
volunteers collected a total of 1,198 M. franciscanus > 40 mm test diameter from urchin
barrens, restoration sites, and kelp reference sites on six sampling dates (29 April, 28 May,
26 June, 22 July, 30 October, and 18 November) throughout 2014. On each collection date,
urchins were collected from one urchin barren, one kelp reference, and two restoration sites
(Fig. 2, Table 1). Divers attempted to collect at least 30 M. franciscanus along a 30m x 2m
transect using pry bars. They were placed into game bags, brought to the boat, and placed
in dry coolers. They were then transported live to Loyola Marymount University’s Seaver
Science Center for dissection. Urchin tests were measured to the nearest mm using calipers
and urchin weight was measured to the nearest hundredth of a gram. Gonads (Fig. 1) were
then removed and weighed to nearest hundredth of gram. All analyses and figures for this
study were produced in R (R Core Development Team 2021).
The modeling approach used in this analysis followed Claisse et al. (2013). An allometry
model was used to quantify the relationship between mean gonad weight and test diameter
length: G = a(L — 40)° (Eq. 1) (Ebert et al. 2011), where G is gonad weight (g), L is test
diameter length (mm), 40 mm is the test diameter length at which M. franciscanus is able
to first produce a gonad and reproduce (Tegner and Dayton 1981; Kato and Schroeter
1985; Tegner 1989), and both a and £ are equation constants. Mean gonad weight at test
diameter length was fitted to the data using the ‘mle2’ package in R by minimizing the
negative log-likelihood and assuming that G follows a lognormal distribution with mean
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URCHIN GONAD RESPONSE TO RESTORATION 7
Apr May Jun Jul
Site Type
= Barren
== Kelp
== Restoration HMC
=e Restoration UAC
Site Name
o HMC
Gonad Weight (g)
=)
o
D
Test
Fig. 3. The relationship between mean gonad weight (g) and test diameter size (mm) of M. franciscanus
collected from April-November 2014 by the site type (color) and site name (shape): Honeymoon Cove
(HMC), Lunada Bay (LB), Underwater Arch Cove (UAC), Marguerite Cove (MC), and Rocky Point (RP).
Parameter estimates for each curve are reported in Appendix Table Al and A2. One point in the November
panel from Restoration HMC (gonad weight 121.52 g at 91 mm test diameter) is not shown so to better
visualize the y-axis range of most data.
determined by Eq. 1 and the standard deviation of the logarithm (sdlog) (Bolker 2008;
Claisse et al. 2013).
In order to account for differences in M. franciscanus size structure among urchin bar-
rens, kelp reference, and restoration sites, a bootstrapping approach was used to estimate
the 95% confidence intervals to compare differences in mean gonad weight at test diameter
length from each site following Haddon (2011) and Claisse et al. (2013). The 95% confi-
dence intervals were then used to assess differences between site types on each collection
date, with non-overlapping 95% confidence intervals considered “significant”. In addition
to the full model, we also specifically compared 95% CIs for mean gonad weight at test
diameter lengths 84 mm and 68 mm. The size of 84 mm was chosen as it is the minimum
size limit of M. franciscanus in the California urchin fishery, while 68 mm was chosen as
being more representative of the size structure within urchin barrens, since most urchins in
these sites were < 84 mm.
Results
Mesocentrotus franciscanus urchin gonad weight at a given test diameter in restoration
sites was higher than in urchin barrens and similar to kelp reference sites throughout most
of the year following the completion of restoration activities (Fig. 3). By May 2014, gonad
production in the sampled restoration sites had “recovered,” i.e., mean gonad weight at a
given test size was similar to that from kelp sites (Fig. 3), and significantly higher than mean
gonad weights at barren sites (Fig. 4, Appendix Table Al, Appendix Fig. A2). A seasonal
temporal pattern in this relationship was also present for each site type with gonad weight
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8 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
68 mm
test diameter
40
30
Site Type
$ Barren
r Kelp
“ Restoration HMC
% Restoration UAC
84mm Site Name
test diameter tf) HMc
40 0) LB
4A wc
> RP
30 | VY vac
J eat MT
T T T T T
Apr May Jun Jul Oct Nov
Collection Date
Gonad Weight (g)
Fig. 4. Mesocentrotus franciscanus mean gonad weight (g) with 95% bootstrap confidence interval error
bars at 68 mm (top) and 84 mm (bottom) test diameter collected from April-November 2014 by the site type
(color) and site name (shape): Honeymoon Cove (HMC), Lunada Bay (LB), Underwater Arch Cove (UAC),
Marguerite Cove (MC), and Rocky Point (RP). The size of 84 mm was chosen as it is the minimum size limit
of M. franciscanus in the California urchin fishery, while 68 mm was chosen as being more representative
of the size structure within urchin barrens, since most urchins in these sites were < 84 mm. Mesocentrotus
franciscanus collected from Rocky Point (kelp reference site) in October 2014 were not included in top panel
because all urchin test diameters exceeded 68 mm. Mesocentrotus franciscanus collected from Underwater
Arch Cove barren sites May-July were not included in the lower panel because all urchin test diameters
were less than 84 mm. Mean gonad weights at test diameter length with 95% CIs are reported in Appendix
Table Al.
at a given length generally increasing across the April to November sampling period (Fig. 3,
Appendix Fig. Al). In April 2014, M. franciscanus gonads from restoration sites had an
average of 72.5% and 60% larger weight at test diameters 68 mm and 84 mm than those
from urchin barrens, respectively. During the peak season (October-November) the average
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URCHIN GONAD RESPONSE TO RESTORATION 9
Apr May Jun Jul Oct Nov
15
ow
10 9
o
5 —
0
15
10 o
ao]
>
3 5
=
o 0
=)
o ®
© 15 ay
= i)
10 o
°
|
5 oa
ss
0 oO
ry)
15 o
S
10- a
3
5 =
>
0 oO
oo o 9°
+t oOo 0 ©
Diameter (mm)
Fig. 5. Mesocentrotus franciscanus test diameter (mm) size distribution (5 mm size classes) for urchins
collected across April to November 2014 from urchin barrens (red), kelp reference sites (green), and the two
restoration sites (blue): Honeymoon Cove (HMC) and Underwater Arch Cove (UAC). Mean lengths are
indicated by a vertical dashed line. All M. franciscanus less than 40 mm test diameter were removed from
analysis, which is the size at which they can first produce a gonad and reproduce (Tegner and Dayton 1981;
Kato and Schroeter 1985; Tegner 1989).
gonad weight at 68 mm and 84 mm test diameter at restoration sites increased to 128% and
154% greater than those from urchin barrens, respectively (Fig. 4, Appendix Table A1).
Generally, M. franciscanus collected at urchin barren sites had smaller test diameters and
those collected at kelp reference sites had larger test diameters, with those from restoration
sites falling in between (Fig. 5, Appendix Table A3). In all six data collection months be-
tween April-November 2014, M. franciscanus collected at kelp reference sites had greater
mean test diameter than those collected at urchin barrens and restoration sites. In five of
the six data collection months, M. franciscanus collected at restoration sites had higher
mean test diameters than those collected at urchin barrens.
Discussion and Conclusions
Reducing S. purpuratus density in urchin barrens on nearshore rocky reefs along the
Palos Verdes Peninsula to the target density of 2/m* through active culling resulted in
the recovery of normal seasonal M. franciscanus gonad production throughout the 8 mos
sampled following completion of restoration activities. Gonad weight at a given length
from urchin in restoration sites matched, and sometimes exceeded, the gonad production
vz0z Jequisoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-pESr-ZOLZYVLOZS6LE/L/L/2z Lpd-sjomesunelinqseos/woo sseidualje uelpuew//:dyy Woy pepeojumoq
10 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
of urchin in kelp reference sites. For M. franciscanus collected throughout 2014, test di-
ameter, gonad weight relative to test diameter, and gonad weight at a given test diameter
in restoration sites were higher than in urchin barrens and similar to those in kelp ref-
erence sites. Even though a seasonal effect of increased gonad production was apparent
at all site types throughout the year, in restoration sites, M. franciscanus gonad weight of
legal-size urchins (84 mm test diameter) were 154% higher than in urchin barrens dur-
ing October and November when the largest gonads weights were observed. Claisse et al.
(2013) estimated the potential effects of restoration on gonad biomass by reporting dif-
ferences between urchin barrens and kelp reference sites indicating restoration could po-
tentially increase gonad biomass available to the commercial urchin fishery. The present
study expands on those findings by also assessing urchins in restoration sites relative to
urchin barrens and kelp reference sites using the same methods. These results build upon
a concurrent study on the Palos Verdes Peninsula, which concluded that declines in urchin
density initiated a quick recovery of kelp dominated state in approximately 6 mos (Williams
et al. 2021), a timeline consistent with the results of other urchin reduction kelp restoration
studies (Sangil and Hernandez 2022).
A seasonal pattern was evident in M. franciscanus as gonad weight relative to test di-
ameter size increased from the earlier to later months in 2014 across all three site types.
This pattern has been observed elsewhere in southern California, as sea urchin spawn-
ing normally occurs in the winter and early spring after an annual peak in gonad size is
reached in the late fall, although there is some variability among geographic areas (Kato
and Schroeter 1985; Ebert et al. 1994; Teck et al. 2018). These seasonal increases in gonad
development coincide with natural patterns of kelp growth and recovery following winter
and spring storms (Cavanaugh et al. 2011; Teck et al. 2018). Spawning then occurs right
after peak gonad production when kelp becomes less abundant (Teck et al. 2018). There-
fore, increases in gonad weight relative to test diameter size at all three site types in our
study was likely due to natural growth of kelp in the summer and fall months. In restora-
tion sites, gonad weight relative to test diameter increased in accordance with this seasonal
pattern but at an even greater magnitude than kelp reference sites and urchin barren sites,
indicative of the additional effect of restoration on gonad production. Interestingly, urchin
barrens also exhibited a low level of seasonal increase on gonad production, even though
these sites maintained an almost complete lack of macroalgae (Williams et al. 2021).
This is expected considering natural increases in macroalgae abundance over this period
(Cavanaugh et al. 2011; Teck et al. 2018) would result in increased drift algae availability
across all site types, in turn, increasing gonad production even at barren sites (Rogers-
Bennett et al. 1995; Britton-Simmons et al. 2012).
Claisse et al. (2013) determined that although M. franciscanus densities were far greater
in urchin barrens than in kelp forests, the lack of gonad production in urchins from barrens
resulted in gonad biomass in kelp reference sites greatly exceeding the overall biomass from
urchin barrens. However, collections from their study were made in April-May 2011, prior
to the peak season of gonad development (Claisse et al. 2013; Teck et al. 2018). When
comparing mean gonad weight at 84 mm test diameter, the minimum size limit for the
fishery, between urchin barrens and restored sites, we found the difference increased from
60% greater in April to 154% greater during the peak season (October-November). This
suggests that the estimates made by Claisse et al. (2013) that restoration could potentially
result in an approximately 900% increase in M. franciscanus gonad biomass available to the
fishery were likely conservative, and the fishery benefits of restoration may be substantially
higher.
vz0z Jequieoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-vESr-ZOLZVLOZ86LE/L/L/2z Lpd-sjomesunslinqseos/woo sseidualje uelpuew//:dyy Wo pepeojumoq
URCHIN GONAD RESPONSE TO RESTORATION 1]
The test size distributions of collected M. franciscanus were significantly smaller at
urchin barren sites than at kelp reference sites, while those in restoration sites generally
fell in between [p < 0.001; mean test diameter (95% CI); Kelp 90 mm (84 — 96), Barren
56 mm (50 — 62), Restoration HMC 75 mm (69 — 81), Restoration UAC 68 mm (62 — 74);
Fig. 5]. These results are consistent with Claisse et al. (2013) who found urchins collected
in kelp reference sites had mean test diameters that were approximately 50% larger than
those in urchin barrens. Similarly, Williams et al. (2021) found that average test lengths of
S. purpuratus were also lower urchin barrens but increased following recovery to kelp forest
conditions.
Resource management strategies that maintain urchin predator abundance, such as ma-
rine protected areas, are beneficial to maintaining stable kelp forests (Kawamata and Taino
2021), however, additional adaptive management actions are likely necessary beyond es-
tablishing marine reserves if urchin barrens are extensive (Levin and Lubchenco 2008;
Baskett and Salomon 2010; Bonaviri et al. 2011; Claisse et al. 2013; Gizzi et al. 2021;
Miller et al. 2022). While marine reserves can increase urchin predator populations such as
the California spiny lobster (Panulirus interruptus) and California Sheephead (Bodianus
pulcher) (Teck et al. 2017), this recovery can take more than fifteen years after imple-
mentation of protection and fishing ceases (Malakhoff and Miller 2021). Further, Eurich
et al. (2014) experimentally demonstrated that California spiny lobster would actively select
S. purpuratus from kelp forests over those from barrens, likely due to their diminished nu-
tritional capacity with a lack of gonad tissue. Accordingly, the recovery of gonad biomass
production observed in our study after culling S. purpuratus should accelerate the return
of these important trophic pathways, ultimately increasing the resilience of restored kelp
forests. Quantifying gonad production in sea urchins is an important measure of ecological
function in kelp forest ecosystems and should be used to inform adaptive management of
restoration projects.
Acknowledgements
This study was supported by the National Oceanic and Atmospheric Administra-
tion Restoration Center, the trustees of the Montrose Settlements Restoration Program,
University of Southern California Sea Grant (Sub-Award No. 162268), and California Sea
Grant (Project No. R/MPA—27A). B. Grime was also supported by a Graduate Student
Research Award from the CSU Council on Ocean Affairs, Science & Technology (COAST)
and received funding from CPP Biological Sciences Department. All project activities were
completed in accordance with the terms of the CDFW Scientific Collecting Permit issued
to the Bay Foundation (TBF; S-183390001-19133-001). We would like to thank all individ-
uals from TBF Marine, Watershed, and Community Engagement Teams who collected
sea urchins, contributed to urchin dissections, and organized volunteer events. Thank
you to all at the Vantuna Research Group for their partnership on this project: Dr. Dan
Pondella, Jonathan Williams, Chelsea Williams, Laurel Zahn, and Matt Robart. Thanks
also to the commercial sea urchin harvesters who worked on clearing urchin barrens,
putting in countless hours of work toward kelp forest restoration.
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vz0z Jequisoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-pESr-ZOLZVLOZ86LE/L/L/2z Lpd-sjomesunelinqseos/woo sseidualje uelpuew//:dyjy Woy pepeojumoq
URCHIN GONAD RESPONSE TO RESTORATION
Appendix Table Al.
Collection
Date
29 Apr 2014
28 May 2014
26 June 2014
22 July 2014
30 Oct 2014
18 Nov 2014
Site
Type
Barren
Kelp
Rest.
Rest.
Barren
Kelp
Rest.
Rest.
Rest.
Rest.
Barren
Kelp
Rest.
Rest.
Barren
Kelp
Rest.
Rest.
Barren
Kelp
Rest.
Rest.
Barren
Kelp
Rest.
Rest.
15
Model parameter estimates of M. franciscanus mean gonad weight at test diame-
ter 84 mm and 68 mm with 95% bootstrap CIs in parentheses for urchins collected April-November 2014
from urchin barrens, kelp reference, and restoration sites (Honeymoon Cove (HMC), Lunada Bay (LB),
Underwater Arch Cove (UAC), Marguerite Cove (MC), Rocky Point (RP)). M. franciscanus collected from
Underwater Arch Cove barren sites May-July were removed from this analysis because all urchin test diam-
eters were less than 84 mm. Mesocentrotus franciscanus collected from the Rocky Point kelp reference site
in October were removed from this analysis because all urchin test diameters exceeded 68 mm.
Site
Name
HMC
LB
HMC
UAC
UAC
LB
HMC
UAC
UAC
HMC
UAC
LB
HMC
UAC
UAC
LB
UAC
HMC
MC
RP
Site ID
UAC JLR
84 mm Test Diameter
Mean Weight (g)
6.9
L339
Pie
10.7
20.8
15,3
15.0
16.3
17.4
95% CI
(5-9.3)
(11-15.9)
(10-12.7)
(7.6-13.9)
(17.8-23.6)
(13.1-17.5)
(13-17.3)
(13.6-19.4)
(15.9-19)
(13.1-17.8)
(20.5-28.3)
(14.1-21.9)
(15.8-20.6)
(12.4-21.3)
(14.6-20.5)
(9.6-17.8)
(17.8-32.4)
(32.8-45.4)
(23.7-38.9)
(10.2-16.1)
(27.8-35.8)
(22.6-32)
(25.1-38)
68 mm Test Diameter
Mean Weight (g)
95% CI
(2.9-4.6)
(3.6-7.8)
(5.1-6.6)
(5.2-8.6)
(0.62.6)
(6.3-10.5)
(7.5-10.3)
(7.9-10.2)
(810.4)
(6.4-9.2)
(1-93-7)
(6.5-9.9)
(9.7-12.8)
(7.1-10.1)
(2.6-4.3)
(5-8.1)
(9.1-13)
(6.1-14.3)
(6.8-10)
(22-26.9)
(16.9-23)
(7.2-10)
(12.7-22.7)
(11.3-15.4)
(15.6-21.6)
vz0z Jequisoeq ¢z uo \senB Aq Jpd"|-1-ZZ1-vESr-ZOLZVLOZ861LE/L/L/2z Lpd-sjomesunelinqseos/woo sseidualje uelpuew//:dyjy Woy pepeojumoq
16 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Appendix Table A2. Model parameter estimates of M. franciscanus mean gonad weight at test diameter
for urchins collected April-November 2014 from urchin barrens, kelp reference, and restoration sites (Hon-
eymoon Cove (HMC), Lunada Bay (LB), Underwater Arch Cove (UAC), Marguerite Cove (MC), Rocky
Point (RP)).
Collection Date Site Type Site Name Site ID a 8 sdlog
29 Apr 2014 Barren HMC HMC_B1_B 0.0400 1.36 0.67
Kelp LB LB_K1_K 0.0136 1.83 0.37
Rest. HMC HMC_T1_R 0.0495 1.44 0.41
Rest. UAC UAC_J1_R 0.2823 0.96 0.86
28 May 2014 Barren UAC UAC_B2_B 0.3416 0.40 0.89
Kelp LB LB_K2_K 0.0106 2.00 0.36
Rest. HMC HMC_T1_R 0.1722 1.19 0.47
Rest. UAC UAC_J1_R 0.2211 1.11 0.48
Rest. UAC UAC_2_R 0.1202 1.30 0.53
Rest. HMC HMC_RIR 0.0170 1.83 0.34
26 June 2014 Barren UAC UAC_B3_B 0.2110 0.75 0.84
Kelp LB LB_K3_K 0.0940 1335 0.45
Rest. HMC HMC_T1_R 0.0369 1.71 0.48
Rest. UAC UAC_J1_R 0.0467 oS 7- 0.55
22 July 2014 Barren UAC UAC_B4_B 0.1876 0.86 0.60
Kelp LB LB_K4 K 0.0021 2.40 0.47
Rest. UAC UAC_J1_R 0.6185 0.86 0.36
Rest. HMC HMC_R1IR 0.6817 0.85 0.60
30 Oct 2014 Barren MC MC_B5_B 0.3675 0.93 0.61
Kelp RP RP_K5_K 0.0344 1.69 0.41
Rest. HMC HMC_T2_R 0.7961 1.03 0.36
Rest. UAC UAC_WI1_R 0.8570 0.94 0.42
18 Nov 2014 Barren MC MC_B6_B 0.4363 0.89 0.56
Kelp LB LB_K6_K 0.3130 1.22 0.53
Rest. HMC HMC_T1I_R 0.0692 1.58 0.54
Rest. UAC UAC_J1_R 0.3722 ies 0.60
vz0z Jaquisoeq ¢z uo jsanB Aq Jpd"|-1-ZZ1-pESr-ZOLZVLOZ861LE/L/L/2z Ljpd-sjomesunelinqseos/woo sseidualje uelpuew//:dyjy Wo pepeojumoq
URCHIN GONAD RESPONSE TO RESTORATION 17
Appendix Table A3. Mesocentrotus franciscanus collection metadata and descriptive summary statistics
for each urchin collections April-November 2014 (Honeymoon Cove (HMC), Lunada Bay (LB), Underwa-
ter Arch Cove (UAC), Marguerite Cove (MC), Rocky Point (RP)).
; ; j Test Diameter (mm) Gonad Weight (g)
Collection Site Site
Date Type Name _ Sub-siteID n Min Med Mean Max SD Min Med Mean Max SD
29 Apr 2014 Barren HMC HMC_BI_B 47 43 56 58 = 84._so11-—S0.00 «1.84 2.58 87.92 2.23
Kelp LB LB_KI_K 30 66 97 OS. JNS- Wis 6/30922,03-23,53 61.01 12.76
Rest. HMC HMC_TIR 49 42 83 76 110 17 0.41 10.48 10.20 25.36 7.27
Rest. UAC UAC JI_R 48 41 = 72 69 102 15 0.15 9.24 8.90 22.98 6.31
28 May 2014 Barren UAC UAC_B2.B 26 41 49 49 72 8 0.00 0.88 1.01 4.15 0.89
Kelp LB LB_K2 K 37 56 96 93 111 15 2.29 35.70 34.78 86.16 19.94
Rest. HMC HMC _TIR 52 52 75 75 94 9 1.79 12.60 12.98 25.97 6.39
Rest. HMC HMC RI R 51 57 87 84 104 12 1.88 17.72 18.82 44.02 10.19
Rest. UAC UAC _JI_R 50 41 = 75 73° 102 17 0.71 11.11 12.88 38.12 10.03
Rest. UAC UAC 2 R 62 45 71 72 100 15 0.67 9.99 12.48 44.64 9.73
26 June 2014 Barren UAC UAC_B3_B 36 41 = 47 51 74 11 0.00 1.00 1.60 8.36 2.06
Kelp LB LB_K3 K 33 45 83 80 104 16 0.71 15.48 15.65 38.47 10.42
Rest. HMC HMC _TIR 38 45 = 72 70 «91 15 0.19 12.19 15.85 52.91 13.66
Rest. UAC UAC _JI_LR 39 44 72 71 98 13 0.64 9.61 12.71 37.60 9.55
22 July 2014 Barren UAC UAC _B4B 29 41 50 SI 73 8 O13 1.54 1.61 4.47 1.15
LB_K4
Kelp LB B_K4 K 50 62 85 85 102 9 2.45 20.20 21.86 61.44 13.24
Rest. HMC HMC RI R 43 41 83 82 120 14 1.36 15.37 19.91 54.10 13.59
Rest. UAC UAC _JI_R 53 42 54 55 82) 9 (0.95 5.26 6.81 28.85 5.18
30 Oct 2014 Barren MC MC_B5S_B 58 41 = 64 63 85 11 0.86 5.68 8.51 29.62 6.87
Kelp RP RP_KS_K 42 79 98 101 123 11 6.48 38.34 39.14 72.82 14.88
Rest. HMC HMC R 47 45 65 64 87 11 4.83 20.03 22.87 60.27 14.11
Rest. UAC UAC_WIR 60 43 61 63 94 12 3.48 12.96 17.85 59.04 13.14
18 Nov 2014 Barren MC MC_B6B 55 41. 60 61 100 13 0.80 6.70 7.37 21.37 5.13
Kelp LB LB_K6 K 54 42° 88 85 119 16 1.08 40.04 37.66 93.68 21.19
Rest. HMC HMC TIR 55 45 79 76 98 14 0.44 18.76 23.67 121.52 20.73
Rest. UAC UAC JI_LR 54 48 78 75 98 12 2.54 27.57 28.18 70.50 16.19
vz0z Jequisoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-pESr-ZOLZVLOZ86LE/L/L/2z Lpd-sjomesunelinqseos/woo sseidualje uelpuew//:dyjy Woy pepeojumoq
18 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Barren Barren Barren Kelp Kelp Restoration Restoration
HMC MC UAC LB RP HMC UAC
125
1005
Collection
3 Date
= 754 ee
Sb ~~
= May
= Jun
3 504 Jul
5
o — Oct
—— Nov
254 i
ofaete™ e mal
S2Q9290092009009000
Appendix Fig. Al. The relationship between gonad weight (g) and test diameter size (mm) of M. fran-
ciscanus collected from April-November 2014 for urchin barrens, kelp reference sites, and restoration sites.
Collection date is designated by color.
Apr May Jun Jul Oct Nov
— =.
Site Type
—— Barren
— Kelp
== Restoration HMC
== Restoration UAC
Gonad Weight (g)
° oh 3 a
T T T T T T T 7 T T T T T T
oo oa oo oo o i=) oO oOo Oo 2 oo o oOo o
owoo nwt O DO CO Oo o ono Oo Tr oOo Oo WN
= r- _— r- r- 1 a - A Sd
Test Diameter (mm)
Appendix Fig. A2. The relationship between gonad weight (g) and test diameter size (mm) of M. fran-
ciscanus collected from April-November 2014, distributed by the site type (urchin barren: red; kelp refer-
ence: green; Honeymoon Cove restoration: light blue; Underwater Arch Cove restoration (dark blue). 95%
bootstrapped confidence intervals are displayed for each curve corresponding to the site type. Parameter
statistics for estimating curves are in Appendix Table 1.
vz0z Jaquieoeq ¢z uo \sanB Aq Jpd"|-1-ZZ1-pESr-ZOLZVLOZ86LE/L/L/2z Ljpd-sjomesunslinqseos/woo sseidualje uelpuew//:dyy Wo pepeojumoq
Bull. Southern California Acad. Sci.
122(1), 2023, pp. 19-32
© Southern California Academy of Sciences, 2023
Developing Growth Promotion Strategies for Cressa truxillensis to
Improve Success of Restoration Activities
Hannah Lyford,!* Michelle R. Lum,!:** Kasra Arjomand,!:? Caroline Ehren,!* and
Karina Johnston?
' Biology Department, Loyola Marymount University, Los Angeles, CA 90045
* Coastal Research Institute, Loyola Marymount University, Los Angeles, CA 90045
3 University of California Santa Barbara, Santa Barbara, CA 93106
Abstract.—Cressa truxillensis, commonly known as alkali weed, is native to west-
ern North America and is used in revegetation projects in saline or alkaline soils at
locations such as the Ballona Wetlands Ecological Reserve. This research aimed to
(1) determine methods to improve C. truxillensis seed germination, (ii) characterize
the impact salt has on seed germination and growth, and (iii) identify and character-
ize bacterial seed endophytes and their potential as plant growth promoting bacteria
(PGPB). Results showed that seed scarification, either through mechanical or chem-
ical methods, substantially improved seed germination rates. The presence of salt at
300 mM NaCl delayed germination, and both 150 mM and 300 mM NaCl decreased
seedling size. Two different strains of Paenibacillus peoriae were found to reside within
C. truxillensis seeds collected from the Ballona Wetlands. Although neither strain alle-
viated the salt sensitivity displayed by C. truxillensis, both strains showed tolerance to
heavy metals, salt, and showed additional properties suggestive that they may function
as PGPB. Methods used in this study can serve as guidelines for preparation of seed of
C. truxillensis prior to seeding in appropriate habitats throughout the species’ range.
Wetlands provide important ecosystem services such as supporting biodiversity, filter-
ing water, sequestering carbon, and protecting against storm surge (Zedler and Kercher
2005; Duarte et al. 2013; Benson et al. 2019). Anthropogenic impacts have resulted in a
global loss of approximately half of the world’s wetlands (Zedler and Kercher 2005). Wet-
land losses in California have exceeded 90% in the last 200 years, thus creating a contin-
ued need for wetland research, conservation, and restoration or enhancement to prevent
and slow further loss (Allen and Feddema 1996). Wetlands have increasingly faced threats
by invasive species, anthropogenic impacts, and changes in the climate; however, restora-
tion by revegetation with native plants provides one possible solution to returning wetland
ecosystem services. Revegetation approaches include transplantation of plants or rhizomes,
planting plugs, and direct seeding (Godefroid et al. 2011; Kettenring and Tarsa 2020).
Understanding the optimal conditions to promote seed germination and plant establish-
ment of target species is of substantial importance to wetland restoration and management.
A seed-based approach to vegetative restoration has the advantage of being less expen-
sive and logistically less challenging to implement (Kettenring and Tarsa 2020). However,
there is a range of soil conditions and hydrology that are likely to affect in situ field ger-
mination (e.g., nutrient availability, soil grain size and porosity, invasive species presence,
etc.), and the downside is that many plant species exhibit extremely low seed germination
* Corresponding author: michelle.lum@Imu.edu
19
z0z Jaquieoeq ¢z uo ysanB Aq Jpd’6-L-ZZL-vESp-Z9LZV/G89861LE/6L/L/Zc LApd-sjoe/uns|inqseos/wioo sseidus|je'uelpuew//:dyjy Wod) pepeojumoq
20 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
rates, especially due to abiotic stresses such as salt or the presence of a hard seed coat that
results in dormancy and prevents imbibition (Almansouri et al. 2001; Baskin and Baskin
2014, 2020). To break dormancy, scarification can break the seed coat and allow a seed to
imbibe and eventually germinate (Baskin and Baskin 2014). A better understanding of ef-
fective scarification techniques for individual species that are targets of restoration projects
is essential for the success of a seed-based approach.
A plant’s tolerance to abiotic stress can also be manipulated, with numerous examples of
plant growth promoting bacteria (PGPB) forming beneficial relationships with host plants
(Cassan et al. 2009; Enebe and Babalola 2018). PGPB can reside within the seed, as well
as on and inside plant roots, promoting plant growth and resilience via biochemical prop-
erties, resistance to disease, and acting as a buffer to abiotic stressors (Eida et al. 2018).
Determining optimal seed germination to account for these inhibitory factors can be im-
portant for seeding plants during restoration, leading to a higher percentage of germinated
seeds. With a growing need for restoration and improving seeding success of suitable plants
for revegetation, this study aimed to inform effective growth strategies of the California na-
tive wetland plant, alkali weed, Cressa truxillensis.
Cressa truxillensis, commonly known as akali weed, is a facultative wetland plant native
to the western United States and commonly found in many southern California wetlands
(Lichvar 2012). Cressa truxillensis is known to have a late spring bloom period with hall-
mark characteristics including a high salinity tolerance and high calcium carbonate tol-
erance (Jepson and Hickman 1993). Studies on C. cretica, another species in the genus,
found that the plant could grow in soils with an upper range of 22% calcium carbonate
and saline concentrations up to 400 mM (Kabir et al. 2010; Sivasankaramoorthy et al.
2011). Although often found in wetlands, the plant can also thrive as a native in many arid
and alkaline habitats, making it an important focus for the conservation or enhancement
of multiple habitat types. The Cressa genus has very low germination rates in its naturally
occurring habitats, which has been attributed to a hard seed coat and suboptimal environ-
mental conditions (Etemadi et al. 2020). This research project aimed to inform the current
state of knowledge regarding the use of this species in revegetation projects by investigat-
ing C. truxillensis scarification techniques and through characterization of its microbial
endophytes.
Materials and Methods
Different scarification methods were applied to seeds of C. truxillensis to determine op-
timal conditions to increase the number of seeds that imbibe and germinate. Seeds for the
scarification experiment were obtained from S&S Seeds (www.ssseeds.com). C. truxillensis
seeds are ovular, brown, and 2-4 mm in length (Austin 1998). Four approaches were taken
to scarify C. truxillensis seeds, including three mechanical and one chemical approach
(Table 1). Each treatment had five replicate batches of 20 seeds. Chemical scarification
was performed by immersing seeds in 0.5 mL of concentrated sulfuric acid in a centrifuge
tube for 45 min. The seeds were then rinsed with sterile distilled water five times to remove
all traces of the acid. The three methods of mechanical scarification were scratching indi-
vidual seeds against sandpaper, rubbing seeds in a batch between sandpaper, and nicking
the seed coat with a razor blade. For scarification of individual seeds by sandpaper, each
seed was scratched across 220-grit sandpaper for 2.5 cm. For the batch treatment, seeds
were randomly dispersed between two pieces of 220-grit sandpaper, a book placed on top
to apply even pressure, and a circular motion applied to rub the seeds between the sheets
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CRESSA TRUXILLENSIS GROWTH PROMOTION STRATEGIES 21
Table 1. Methods for scarification of C. truxillensis seeds
Chemical Mechanical
Scarification Type Acid Individual Batch Razor Blade
Description Seeds immersed for Seeds individually Seeds sandwiched — Seed coat nicked
45 minin sulfuric — scratched on between (Mackay et al.
acid (Etemadi sandpaper sandpaper, 1996).
et al. 2020). (Hassen et al. scratched (Hassen
2005). et al. 2005).
of sandpaper for 10 passes. To scarify seeds with a razor blade, each seed was nicked once
to break the surrounding seed coat. Batches of untreated seeds were placed in petri dishes
to serve as controls. For all treatments and the controls, seeds were placed in autoclaved
petri dishes containing filter paper saturated with 7 mL of sterile deionized water. The petri
dishes with seeds were wrapped in parafilm to prevent evaporation and then placed in the
dark at 24°C. The number of germinated seeds within each dish was counted after seven
days.
Bacteria associated with C. truxillensis were identified, as many plant-associated bac-
teria have plant-growth promoting properties. C. truxillensis seeds were collected from
the ground, within C. truxillensis and Salicornia pacifica plant communities in the non-
tidal high salt marsh and transition habitats of the western area B marsh at the Ballona
Wetlands Ecological Reserve (Los Angeles, CA, 33.963025°, -118.447015°). No more than
10% of available seed was collected from within individual polygons that were identified
throughout the appropriate habitat areas, ensuring that no seed banks were impacted dur-
ing the collection. A total of 150 seeds were collected from the Ballona Reserve. Seeds were
scarified and surface sterilized with 95% ethanol for five minutes, followed by 10% bleach
for 10 min, and then rinsed with five washes of sterile deionized water. The water from
each wash step was plated onto Tryptone Yeast extract agar (TY; 5 g/L tryptone, 3 g/L
yeast extract, 0.0662 g/L CaCl-2H,0, 15 g/L agar) and incubated at 30°C to rule out
contamination by a lack of microbial growth on the media. Following sterilization, seeds
were crushed individually in 5 mL of sterile saline (0.9% NaCl) using a sterile mortar and
pestle. Dilutions of the bacterial suspensions were plated onto TY agar and incubated at
30°C (Siddikee et al. 2010). Dilutions were made so that individual colonies would be easily
identified and isolated when grown on TY agar. Two individual bacterial strains, C15 and
C3G, were selected to represent the different colony morphologies seen on Yeast extract
Mannitol agar (YM; 0.5 g/L yeast extract, 10 g/L mannitol, 0.5 g/L KH2POxq, 0.5 g/L
K»HPOsg, 0.2 g/L MgSO,4-7H20, 0.2g/L NaCl, 15 g/L agar). A simple stain with safranin
and a spore stain were performed on each strain and observed by brightfield microscopy
(Zeiss Axioscope 5).
The bacterial strains were characterized for biochemical properties associated with plant
growth promotion and their ability to tolerate salt and heavy metal stress. Strains were
tested for nitrogen-fixation by growth on nitrogen-free Jensen’s media (Jensen 1942) af-
ter 7 d. Cellulase activity was tested by growing the strains on Carboxymethyl Cellulose
(CMC) agar (Kasana et al. 2008), flooding the plates with Gram’s iodine, and checking for
the presence of clear halos around the streaked bacteria. The production of exopolysac-
charide was determined by growing bacteria on YM agar and visually identifying the
presence of a mucoid colony morphology. Phosphate solubilization was tested by visually
z0z Jaquisoeq ¢z uo ysanB Aq Jpd’6-L-ZZL-vESp-Z9LZV/SG89861LE/6L/L/Zc LApd-sjoe/uns|inqseos/wioo sseidus|je'uelpuew//:dyjy Wold papeojumoq
22 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
identifying the presence of clear halos around bacteria on Pikovskaya’s agar (Pikovskaya
et al. 1948) after 7 d of growth. Auxin production was determined by culturing bacteria
in TY supplemented with 1 mg/ml tryptophan and adding Salkowski reagent (Ehmann
1977; Gordon and Weber 1951) after growth. The presence of red coloration indicated the
production of indole acetic acid (IAA). For analysis of bacterial tolerance to abiotic stres-
sors, bacteria were streaked on TY agar that had been supplemented with zinc (50, 100,
200, 400, 750, or 1000 1M ZnSOz), cadmium (50, 100, or 200 pjM CdCl,) or salt (1%, 2%,
3%, 4%, or 5% NaCl), incubated for seven days at 30°C, and growth compared to that seen
on TY. All assays were done in triplicate.
To identify bacterial strains C3G and C15, DNA was obtained from each strain using
a Chelex extraction (Walsh et al. 2013). The DNA was used to amplify the 16S rRNA
gene by the polymerase chain reaction using universal primers 27F and 1492R (Weisburg
et al. 1991). The PCR products were sequenced, and the sequences were compared to that
of bacterial type strains using tools of the Ribosomal Database Project (Cole et al. 2014)
and NCBI BLAST (Altschul et al. 1990). Closely related sequences were retrieved from
the GenBank database. A phylogenetic tree of the 16S rRNA gene was generated using
MEGAX (Kumar et al. 2018; Stecher et al. 2020) and the maximum-likelihood algorithm.
Bootstrap analysis was done for statistical support using 1000 re-samplings with gamma
distribution (G) and the Tamura 3-parameter (T92) model (Tamura 1992). The following
new sequences were deposited in the GenBank database: 16S rDNA of Paenibacillus sp.
strain C15 (OK178930) and Paenibacillus sp. strain C3G (OK178931).
For the seed germination assays including salt stress and bacterial inoculation, C. trux-
illensis seeds were scarified and surface sterilized as described above. Bacterial strains C3G
and C15 were grown overnight in TY media, centrifuged and the bacterial pellet washed
once with 10 mM MgSO, to remove any residual TY, and then centrifuged again and the
bacteria resuspended in 10 mM MgSO, to an absorbance at 600 nm of 0.1 (Montanez et al.
2012). C. truxillensis seeds were incubated for 1 hr in the bacterial suspension or 10 mM
MgSO, as a control. The 35 seeds were transferred in triplicate to a petri-dishes contain-
ing 0.8% agar (Sigma A1296) with either 0, 150, or 300 mM NaCl. The number of seeds
germinated were counted after 7, 14, and 21 days. Wet weight of germinated seedlings was
taken at 21 d.
Results
Cressa truxillensis control seeds exhibited a low average percentage of germination when
seeds were not scarified (5.8% + 3.4%) (Fig. 1). Chemical scarification in concentrated sul-
furic acid for 45 min resulted in the greatest average germination (54% + 6.1%), substan-
tially higher than the unscarified control, although mechanical scarification of individual
seeds via sandpaper (34% + 9.8%) and nicking with a razor blade (29% + 3.4%) also im-
proved average germination (Fig. 1). However, batch scarification did not show substantial
improvement in the percentage of germination (11% + 6.1%) as compared to the control
treatments.
Results indicated that the low salinity treatment (150 mM NaCl) had no impact on per-
cent seed germination of C. truxillensis at 7 days (61% + 1.2%) as it was comparable to
the control (59% + 5.1%) (Fig. 2). However, the high salinity treatment (300 mM NaCl)
caused a reduction in germination (8% + 0.7%) (Fig. 2). By 21 d the percentage of germi-
nated seeds did not vary between saline treatments (Fig. 2). Although the final germination
percentages were similar, seedlings exposed for 21 d to 150 mM or 300 mM NaCl showed
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CRESSA TRUXILLENSIS GROWTH PROMOTION STRATEGIES 23
20 100
18 90
16 80 i
3 Oo
D 14 70 @
Ww) WwW
il
o 12 60 ov
o fae]
€ 10 50 ©
E 8 40 E
<0) <b)
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e 6 30 6
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4 0 >
2 10
0 0
Control Sulfuric Acid Sandpaper- Sandpaper- Razor Blade
Individual Batch
Fig. 1. Effect of four seed scarification treatments on the number of germinated C. truxillensis seeds
(left Y-axis) and the percentage of germinated C. truxillensis seeds (right Y-axis). Values are the mean of
five replicates (20 seeds each replicate) and vertical bars indicate standard error. Different letters indicate a
statistical difference based on One-Way ANOVA with post-hoc Tukey (p < 0.05).
a reduction in growth compared to unexposed seedlings (Fig. 3A and 3B). In addition,
the higher the concentration of salt, the greater the reduction in seedling size and mass
(Fig. 3A and 3B).
Bacteria were isolated from within C. truxillensis seeds that had been collected from the
Ballona Wetlands. Although bacterial colonies looked similar on TY, when transferred to
YM agar they showed two distinct colony morphologies. Strains C15 and C3G were cho-
sen as representatives of each colony type (Fig. 4A and 4B). Most notably, the texture was a
differentiating characteristic between strains C3G and C15; on YM agar, strain C15 had a
mucoid appearance indicating the presence of exopolysaccharide while strain C3G did not
(Fig. 4B). Microscopy of stained bacteria showed that both strains looked similar and were
bacillus in shape (Fig. 4C and 4D). In addition, both strains were found to produce en-
dospores (Table 2). The 16S rRNA gene sequences of strains C3G and C15 were 100% iden-
tical to each other and identified them as species of Paenibacillus, with 99.71% (1393/1397
n.t.) identity to and phylogenetically grouping with P peoriae DSM8320 (Fig. 5).
Table 2._ Biochemical characteristics of endophytic bacteria of C. truxillensis seeds.
Tolerance to abiotic stress PGPB properties
Strain ZnSO, CdCl, NaCl(%) N-fixation Cellulase IAA Phosphate Endospore
C3G 400 4M - 4% + . - + +
CIS ImM 50uM 4% zu rf ie a +
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24 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
35 100
G7 days
a W 14 days
M2idays p
# of Germinated Seeds
3
% of Germinated Seeds
CTR C3G C15 CTR C3G C15 CTR C3G C15
0 mM NaCl 150 mM NaCl 300 mM NaCl
Fig. 2. Effect of NaCl and bacterial inoculation on the number of germinated C. truxillensis seeds
(left Y-axis) and the percentage of germinated C. truxillensis seeds (right Y-axis) over time. Values are the
mean of three replicates (35 seeds each replicate) and vertical bars indicate standard error. Different letters
indicate a statistical difference based on One-Way ANOVA with post-hoc Tukey (p < 0.05). CTR indicates
uninoculated control treatments.
Strains C15 and C3G were tested for biochemical properties associated with plant
growth-promoting bacteria. Both strains grew on nitrogen-free media (Table 2), indicating
that they were able to fix nitrogen, similar to many previously characterized Paenibacillus
strains (von der Weid et al. 2002). In addition, both strains had cellulase activity, produced
auxin, and solubilized phosphate (Table 2). Strains C15 and C3G were tested for growth
at varying concentrations of zinc, cadmium, and sodium chloride in order to look at the
impact of various abiotic stressors. C3G grew with up to 400 ~M ZnSO,, however C15
grew even in the presence of 1 mM ZnSO, (Table 2). The strains also showed varying tol-
erance to cadmium. C3G was sensitive to cadmium, but C15 grew in up to 50 wM CdCl,
(Table 2), highlighting the higher tolerance C15 has to heavy metals. Both strains behaved
similarly to salt stress and could grow in as much as 4% NaCl but were inhibited at 5%
NaCl (Table 2). When C. truxillensis seeds were inoculated with either strain of bacterium,
no difference was seen in the percentage of seeds that germinated, whether or not salt was
present (150 and 300 mM) (Fig. 2). However, in the absence of salt, inoculation with C3G
resulted in a slight increase in seedling wet weight (Fig. 3B).
Discussion
The seed coat can be a major barrier to germination for many plants. How to overcome
this seed coat-imposed dormancy, or “hardseededness”, and thus increase the seed’s per-
meability to water, has been the basis of numerous studies (Tadros et al. 2011; Mousavi
z0z Jaquisoeq ¢z uo ysanB Aq Jpd’6L-L-ZZL-vESp-Z9LZV/S89861LE/6L/L/Zc LApd-sjoe/uns|inqseos/wioo sseidus|je'uelpuew//:dyy Wod pepeojumoq
CRESSA TRUXILLENSIS GROWTH PROMOTION STRATEGIES 25
A.
Seedling Wet Weight (mg)
OmM 150 mM 300 mM
Fig. 3. Effect of NaCl and bacterial inoculation on C. truxillensis seedlings. A) Seedlings after 21 d of
growth with 0, 150, or 300 mM NaCl. B) Average wet weight of seedlings after 21 d of growth on different
concentrations of NaCl. Data represents the mean seedling wet weight from 30 seedlings from three inde-
pendent replicates, and vertical bars indicate standard error. Different letters indicate a statistical difference
based on One-Way ANOVA with post-hoc Tukey (p < 0.05).
et al. 2011). Typical scarification strategies include hot water treatment, chemical scarifica-
tion with acid, or mechanical scarification to nick the seed coat (Rusdy 2017). Though
differing in method, chemical and mechanical scarification work to obtain similar re-
sults of breaking the seed coat. Mechanical scarification may require more labor on fewer
seeds via physical removal or permeation of the barrier, while chemical scarification is
effective for larger batches and uses concentrated acid to dissolve part of the seed coat
(Majd et al. 2013). The use of sulphuric acid to overcome germination difficulties in
seeds has been well-established in the literature as a highly effective scarification technique
(Kheloufi et al. 2017). Congruent with studies of C. cretica, which found that naturally
occurring seeds have very low germination rates (Etemadi et al. 2020), the non-scarified
control C. truxillensis seeds in this study showed a low percentage of germination. This
suggests that restoration or revegetation projects seeding with non-scarified seeds may ex-
perience low initial germination rates due to seed coat-imposed dormancy, which would
delay bringing back native plant cover to an area. An experimental study at the Univer-
sity of Wisconsin comparing hand seeding and transplantation as restoration techniques
found hand seeding to be more successful in producing greater diversity and growth of
z0z Jaquisoeq ¢z uo ysanB Aq Jpd’6-L-ZZL-vESp-Z9LZ/SG89861LE/6L/L/Zc LApd-sjoe/uns|inqseos/wioo sseidus|je'uelpuew//:dyy Wod papeojumoq
26 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Fig. 4. Colony and cellular morphology of bacterial isolates C3G and C15. Bacterial growth on (A)
TY and (B) YM agar. Cellular morphology after staining with safranin of (C) C3G and (D) C15. Arrow
points to mucoid colonies of C15.
C3G (0K178931)
C15 (0K178930)
Paenibacillus peoriae DSM 8320' (AJ320494)
Paenibacillus kribbensis AM49* (AF391123)
Paenibacillus jamilae CECT 5266 ' (AJ271157)
Paenibacillus polymyxa |AM 134197 (D16276)
Paenibacillus brasilensis AF273740 ' (AF273740)
Paenibacillus terrae AM141 ' (AF391124)
100 - Paenibacillus hunanensis FeLO5 ' (EU741036)
Paenibacillus wenxiniae 373 ' (KP212687)
Bacillus subtilis DSM10' (AJ276351)
90
79
99
a
0.050
Fig. 5. Maximum-likelihood phylogenetic tree of the 16S rRNA gene sequences from C. truxillensis
endophytic strains, C15 and C3G, and closely related Paenibacillus type strains. Genbank accession numbers
are in parentheses. Bootstrap values greater than 70% are shown in the nodes. Bacillus subtilis was used as
the outgroup.
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CRESSA TRUXILLENSIS GROWTH PROMOTION STRATEGIES Ox
native plants in addition to greater cost and time effectiveness; however, they noted that
an extended dormant period for unscarified seeds may present problems with the regrowth
of non-native plants (Weiher et al. 2003). Results from this study indicated both mechan-
ical and chemical scarification methods improved C. truxillensis seed germination, show-
ing that scarification is a useful strategy to overcome dormancy. The sulfuric acid scari-
fication technique, which was also effective in scarifying C. cretica (Etemadi et al. 2020),
substantially improved germination rates of C. truxillensis compared to other treatments.
Additionally, the sulfuric acid scarification method allowed for many seeds to be scari-
fied at the same time in a batch, meaning this technique could be a feasible option for
preparing large quantities of seeds prior to seeding for restoration or revegetation ac-
tivities, particularly because of the reduced time and effort. If the percentage of seeds
that are germinating at each restoration site can be increased using pre-scarified seeds,
restoration projects may be able to see more rapid success in reaching native plant cover
objectives.
Bacterial endophytes reside intracellularly or intercellularly within the plant, often act-
ing as beneficial PGPB and helping the plant survive abiotic stress (Rashid et al. 2012;
Egamberdieva et al. 2017). An increasing number of studies have shown bacteria residing
as endophytes in seeds as well (Li et al. 2019; Khalaf and Raizada 2016), with these bacteria
likely derived from the parent plant and contributing to increased fitness and survival of the
seed in its environment (Li et al. 2019; Mastretta et al. 2009; Zhang et al. 2010). Paenibacil-
lus sp. are known to be PGPB for numerous plant species and have been found residing
as seed and root endophytes (Eida et al. 2018; Khalaf and Raizada 2016). Paenibacillus
has been studied in Arabidopsis thaliana and found to protect the plant from abiotic stres-
sors and plant pathogens (Hong et al. 2016). Further studies have tagged the bacteria with
green fluorescent protein and found that the bacteria form biofilms and colonize root tips
of plants (Timmusk et al. 2005). The C. truxillensis bacterial strains isolated in this study
were identical to each other in 16S rRNA gene sequence and were most similar to Paeni-
bacillus peoriae. Although the strains showed variation in exopolysaccharide production
and tolerance to cadmium, they both showed plant growth promoting traits such as nitro-
gen fixation, cellulase production, phosphate solubilization, and auxin production. Con-
sistent with this, after inoculation of C. truxillensis seeds, one of the strains resulted in an
enhancement of seedling mass. In addition, Paenibacillus produce endospores. Bacterial
endospores are a dormant form of bacteria that can withstand harsh chemical and ther-
mal environments and allow the bacteria to survive stressful conditions, thus making them
ideal for inoculant formulations. If these Paenibacillus strains were to be used for plant
growth promotion in the field, their endospores could be explored as a form of inoculant
as this could increase inoculant shelf life due to the ability of endospores to survive variable
conditions (Kim et al. 2010).
Studies have shown that plant associated bacteria, including strains of Paenibacillus, can
help plants better cope with heavy metal stress (Kumari and Thakur 2018; Sukweenadhi
et al. 2018; Eida et al. 2020). The Paenibacillus strains from this study showed tolerance
to zinc and cadmium. Previous studies in the Ballona Reserve showed that zinc and cad-
mium concentrations are relatively high, presumably due to urban runoff and other an-
thropogenic impacts (Johnston et al. 2012). Thus, it is not surprising that bacterial strains
isolated from the Ballona Wetlands have increased tolerance to these heavy metals. A study
of C. truxillensis at a salt marsh in Patagonia, Argentina, found that C. truxillensis was sen-
sitive to heavy metals, with morphology of the leaves altered depending on concentrations
of pollutants such as zinc (Pollicelli et al. 2018).
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28 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Halophytes, which include C. truxillensis, are plants that can tolerate NaCl levels greater
than 200 mM (1.17%). This is consistent with the study findings for C. truxillensis, which
could germinate with at least 300 mM NaCl. This finding, as well as the decreased seedling
growth seen with increasing salt concentrations, is analogous to what has been reported
for C. cretica (Etemadi et al. 2020). Recent studies suggest that plant-associated microbes
can play a key role in the ability of halophytes to grow in high salinity (Etesami and Beattie
2018) but may also help non-halophytes tolerate salt stress. Paenibacillus sp. JZ16, isolated
from inside the roots of the halophyte Zygophyllum simplex, was found to promote salinity
tolerance of Arabidopsis (Eida et al. 2018). The Paenibacillus strains from this study grew
in both the absence of salt as well as in up to 4% NaCl. Both strains also produced auxin,
a trait frequently associated with salt tolerant PGPB (Dodd et al. 2010). It is thought
that the altered hormonal signaling, which can influence lateral root development (Yang
et al. 2009), contributes to the ability of these bacteria to increase the plant’s fitness in
highly saline environments (Siddikee et al. 2010; Tiwari et al. 2011). Other studies have
found that exopolysaccharide may allow bacteria to promote plant growth under saline
stress (Abbas et al. 2019). Since C. truxillensis is known to grow in saline soils, inoculation
with an exopolysaccharide producing strain may allow for optimized plant growth in high
saline conditions. However, although one of the Paenibacillus strains in this study showed
some growth promotion of seedlings in the absence of salt, neither strain, whether or not it
produced exopolysaccharide, alleviated the negative impact salt had on seed germination
or growth under the laboratory conditions tested. Future studies might look at whether
these bacterial strains have a beneficial impact on plant growth with salinity stress using
greenhouse and field conditions.
Many studies have focused on the use of PGPB in agriculture; however, few studies
have explored applications of the microbial community on restoration and native plant
revegetation projects (Ahn et al. 2007). The application and results of this study could in-
form habitat restoration projects throughout the range of C. truxillensis, including at the
Ballona Wetlands. Wetland restoration projects that have focused on returning native cover
to an area have quickly discovered a vast lack of available peer reviewed literature when
it comes to best practices for species-specific cleaning, storage, and breaking dormancy,
despite breaking seed dormancy being a widely known requirement for revegetation
(Kettenring and Tarsa 2020; Barton et al. 2016). Scarification techniques recommended
by this study could be used by practitioners working on habitat restoration projects
with the goal of improving native plant cover. Past restoration efforts within the Ballona
Wetlands have focused on reducing anthropogenic uses and removal of invasive plants
within the area (Johnston et al. 2021); however, the findings from this study could allow for
a unique opportunity to combine knowledge of revegetation techniques with the microbial
community for successful revegetation with C. truxillensis. Application of these findings at
a restoration project would allow for a greater knowledge base around the use of PGPB
for revegetation, as well as development of best practices for germinating wild seeds.
Utilizing the findings from this study, a few key recommendations can be made for fu-
ture revegetation and restoration projects. Regarding improving germination, sulfuric acid
scarification should be used on seeds prior to deployment: this method resulted in the
greatest average percent germination and would also allow for large batches of seeds to be
scarified together, reducing energy and time. Seeds can be inoculated with PGPB’s prior
to seeding, such as the ones identified in this study, to increase plants’ protections against
heavy metals and provide biochemical advantages. Finally, considerations should be taken
to deploy seeds in habitats that include some freshwater hydrology, especially in salt marsh
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CRESSA TRUXILLENSIS GROWTH PROMOTION STRATEGIES 29
soils. Findings suggested that high salinity had a delaying effect on germination of C. trux-
illensis seeds, and freshwater sources may ameliorate this delay from salt stress to provide
maximum germination.
Future directions with these findings will aim to test scarification methods and microbial
applications in situ for applications within restoration projects. Scarification techniques
from this study should also be explored for other native plants commonly seeded in restora-
tion and revegetation projects, including rare species. To further contribute to the available
knowledge informing habitat restorations, pre-scarified seeds could be dispersed within an
area to measure the success and feasibility of scarification methods in varying field con-
ditions. Based on findings in the Pollicelli et al. (2018) study that C. truxillensis has sensi-
tivity to heavy metals, future studies might look at whether the Paenibacillus strains from
this study are able to increase the tolerance of C. truxillensis to certain heavy metals. The
endophytic nature and presence of plant growth promoting traits of the two strains iso-
lated in this study suggest they are PGPB, although the strains did not alleviate the impact
of salt stress under the conditions tested in this study. Future studies can further assess
the conditions in which these strains might promote plant growth and assist C. truxillensis
withstand abiotic stress, and if this would be helpful when planting seeds collected from
other locations that might not carry the same microbes. Formulating inoculants for the
plants and measuring growth could give definitive answers on how well each strain is able
to promote plant growth and other characteristics that may be advantageous for future
restoration projects.
Acknowledgements
This research was supported in part by the William F. McLaughlin Chair in Biology to
M.R.L. H.L., K.A., and C.E. received support through Loyola Marymount University’s
Coastal Research Institute and a grant from the U.S. Environmental Protection Agency.
The authors would also like to thank The Bay Foundation and California Department of
Fish and Wildlife for support.
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Bull. Southern California Acad. Sci.
122(1), 2023, pp. 33-50
© Southern California Academy of Sciences, 2023
Knowledge Gaps and Research Priorities in Living Shorelines
Science: Insights from Stakeholder Interviews Throughout the U.S.
Pacific Coast
Marjorie E. Mednikova,!** Christine R. Whitcraft,” Danielle Zacherl,* and
Kathryn D. Nichols*
! University of New Hampshire, 105 Main St, Durham, NH 03824
* California State University Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840
3 Department of Biological Science, California State University Fullerton, CA 92831
4 Orange County Coastkeeper, F#110, 3151 Airway Ave, Costa Mesa, CA 92626
Abstract.—Living shorelines provide a nature-based strategy for coastal restoration
with ample opportunity for community engagement and collaboration with interdis-
ciplinary stakeholders. While their implementation has increased over the past few
decades, restoration via this technique is limited by several factors including a lack of
data sharing among projects and geographical regions, a shortage of long-term moni-
toring to demonstrate efficacy at meeting project goals, and a need for greater interdis-
ciplinary communication moving forward. In this study, we reviewed recent literature
from a range of living shorelines studies throughout the United States and conducted
interviews with nature-based coastal restoration practitioners primarily from the U.S.
west coast. The insight from these stakeholder interviews allowed us to identify major
knowledge gaps about living shorelines and establish priorities for future research and
funding, including: (1) funding demonstration projects in their early research stages,
(2) supporting projects and trainings for engineers utilizing nature-based infrastruc-
ture, (3) conducting long-term monitoring of both ecological and structural proper-
ties, (4) communicating findings, importance, and project visualizations to stakehold-
ers within and between communities, and (5) advancing the causes of environmental
justice and equity. By reviewing recent literature and engaging with living shoreline
practitioners to gather their experiences and suggestions, we have increased under-
standing of how living shoreline restoration can be more effectively planned, con-
structed, and monitored at scale, in varied locations and using a range of techniques.
Living shorelines restoration is an increasingly important method of enhancing coastal
resilience in the face of changing climate and rising sea levels, as it provides nature-based
alternatives to shoreline armoring while maintaining biodiversity (Bilkovic et al. 2016;
Gittman et al. 2016b; Smith et al. 2020). Coastal restoration using natural infrastructure
furthermore presents opportunities for community engagement and collaboration among
diverse groups of stakeholders (Bragg et al. 2021; Molino et al. 2020; Smith et al. 2020).
These benefits to local ecosystems and communities demonstrate the value of utilizing
living shorelines for coastal resilience on a larger scale. In order to broaden the use of liv-
ing shorelines, however, several limitations to their widescale implementation must first be
addressed.
* Corresponding author: marjorie.mednikova@gmail.com
33
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34 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
First, a consistent definition of living shorelines is lacking; many types of coastal habitat
restoration in different geographic locations are encompassed by the term “living shore-
lines” (Moosavi 2017; O’Donnell 2017; Smith et al. 2020). Furthermore, living shorelines
are highly context-dependent, and extending their use to new coastal communities requires
careful consideration of regional needs and management concerns. Compilations of case
studies varying by landscape setting, energy level, and habitat type are a work in progress,
but a more concentrated effort in developing these resources is needed (Beagle et al. 2019;
SCC et al. 2010; Judge et al. 2017). The concept of using living shorelines 1s still relatively
new, particularly on the Pacific coast of the United States (Bilkovic et al. 2016; Boudreau
et al. 2018; Smith et al. 2020). Restoration designs on the U.S. west coast therefore of-
ten draw from East coast case studies and need to be adapted to Pacific shorelines, which
are characterized by more open coast area and shoreline hardening (Boudreau et al. 2018;
Gittman et al. 2015; Hanak and Moreno 2012). Demonstration projects testing new tech-
niques and evaluating the likelihood of their success, especially along the Pacific coast,
are limited (SCC et al. 2010; Russell and Griggs 2012; Saleh and Weinstein 2016). A cen-
tral goal of our research was to investigate regional differences among approaches to living
shorelines, highlighting projects on the Pacific coast through stakeholder interviews to gain
a better understanding of the challenges specific to this region.
One particular area that merits attention is discussion with stakeholders about successes,
perceptions, and limitations to implementing living shorelines restorations. Several stud-
ies have compiled information on different restoration methodologies through discussion
with experts and literature review to identify restoration goals and guidelines (Baggett
et al. 2015; Fitzsimons et al. 2020; O’Donnell 2017; Smith et al. 2020; Ridlon et al. 2021;
Waltham et al. 2021; Zeigler et al. 2021). These studies have highlighted the need for greater
discussion and data sharing among coastal restoration practitioners. Some research fur-
thermore included a practitioner interview or survey component (Fitzsimons et al. 2020;
Molino et al. 2020; Smith et al. 2020); however, direct interviews with interdisciplinary
stakeholders have a very limited representation in living shorelines literature. Through both
a literature review and interviews with coastal restoration practitioners from a variety of
disciplines and communities, we identify scientific knowledge gaps, priorities for future
research and opportunities for community engagement.
Materials and Methods
The first component of our study consisted of a review of relevant literature encom-
passing diverse perspectives in living shorelines science. The databases Academic Search
Complete, ScienceDirect, Web of Science, and JSTOR were used in addition to Google
Scholar to find peer-reviewed research articles on living shorelines science, from foun-
dational discussions to recent studies incorporating novel methodologies. Several search
terms were used to find literature, given the inconsistencies in living shorelines definitions
and the variety of methods used by practitioners. “Living Shorelines,” “Nature-Based
Infrastructure,’ “Nature-Based Engineering,” “Coastal Resilience,’ “Shoreline Harden-
ing,’ “Coastal Restoration,’ “Coastal Management,” “Oyster Seagrass Restoration,”
and “Multi-Habitat Coastal Restoration” were all searched in each database to find
relevant literature, with secondary terms added including “stakeholders,” “interviews,”
“practitioners,” “community,” and “community engagement.” In addition to the jour-
nal articles resulting from these searches, we furthermore reviewed publications from
nonprofit organizations (e.g., The Nature Conservancy), state and local government
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 35
Table 1. Overview of the distribution, disciplines and affiliations of stakeholders interviewed between
March and June of 2020.
Category Stakeholders interviewed
Geographic region
Northern California 7 (30%)
Southern California 12 (52%)
Washington State 1 (4%)
Hawaii 1 (4%)
East Coast 1 (4%)
Gulf Coast 1 (4%)
Discipline
Biologist 6 (26%)
Oceanographer 1 (4%)
Engineer 4 (17%)
Policymaker 2 (9%)
Funding Work 2 (9%)
Geomorphologist 1 (4%)
Coastal Manager 3 (13%)
Affiliation
Engineering or Consulting Firm 5 (22%)
Federal Agency 1 (4%)
Funding Organization 1 (4%)
Grassroots Initiative 1 (4%)
NGO 2 (9%)
Nonprofit 3 (13%)
Research Institute 1 (4%)
State Agency 3 (13%)
University/ Academia 6 (26%)
agencies (e.g., the California State Coastal Conservancy), white papers, proceedings from
academic society and local government meetings, and mission statements from funding
organizations.
The second component of this study consisted of direct interviews with stakeholders
involved in living shorelines restoration efforts, also referred to in this study as practi-
tioners to reflect their active involvement with restoration projects. These living shorelines
practitioners were selected with the goal of equally representing different disciplines and
organization types along the Pacific coast. Some interviewees based in the East and Gulf
coasts were included due to their familiarity with projects in the Pacific, or their special-
ized knowledge of techniques that could be applied to Pacific coast systems in the fu-
ture. A list of potential contacts was created throughout the literature review process and
was expanded through input solicited from several experts in the field of coastal restora-
tion. Interviewees were selected from nonprofit and grassroots organizations, state and
federal agencies, academia, consulting and engineering firms, and funding groups. Biolo-
gists, ecologists, oceanographers, engineers, policy specialists, and stakeholder engagement
professionals all participated in interviews. Please refer to Table 1 for an overview of the
organization types and disciplines represented among interviewees, in addition to their ge-
ographic distribution.
Interviewees took part in a phone or video call ranging from 25 mins to 1.5 hrs in length
to discuss challenges and opportunities in living shorelines science. The following questions
were asked to each practitioner:
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36 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
1. What have you found to be the biggest challenges or barriers in advancing living
shorelines research in your work?
2. What projects and programs do you think are most needed to help advance living
shorelines and on-the-ground climate resilience measures?
3. What research topics or innovative restoration methodologies would you be inter-
ested in learning more about, both in your community and in other regions? Are
there specific novel techniques for increasing coastal climate resilience that you
think need more support or attention?
4. What are the needs to help enhance scalability of living shorelines projects beyond
individual locations?
5. How is your living shorelines research being used by practitioners and communi-
cated to the public, and how might this be improved upon?
6. Are you seeking any new partnerships or methods of community engagement that
would be particularly valuable in accomplishing your project goals?
7. How can communication around advancements in living shorelines science and
solutions be enhanced within the scientific community?
A total of 23 living shorelines practitioners participated in interviews between March
and June 2020. While 34 practitioners were identified and contacted for interviews based on
our literature review and additional input from experts, the 23 interviewees described here
represent the subset of practitioners contacted who were responsive to our request and able
to participate. Interviews were conducted between March and June 2020. After interviews
were completed, individual responses to interview questions were reviewed and common
themes were identified. The major challenges and opportunities discussed by practitioners
were then compared to those documented in the literature.
Results
Our literature review included a total of 50 sources, incorporating perspectives on living
shorelines projects from the East, West, and Gulf coasts of the U.S. (Fig. 1). The sources
reviewed included journal articles on small-scale living shorelines projects monitored pri-
marily by academic researchers, as well as case study compilations for larger collaborative
efforts between nonprofits and agencies at the State or Federal level. Pacific coast studies
were more limited in scope; of the 50 sources reviewed, only 18 (36%) were specific to the
Pacific coast. Among Pacific coast studies, projects were clustered in areas with low-energy
back bays, including San Francisco Bay and Elkhorn Slough in northern California; New-
port Bay and San Diego in southern California; and Puget Sound in Washington State. The
geographic distribution of sources highlighted the relative lack of literature and case stud-
ies from West coast systems, a result of living restoration techniques being relatively newer
to this region. Comparing sources from both regions allowed us to distinguish knowledge
gaps unique to the Pacific coast from those that are overarching needs in living shorelines
science.
The literature review identified a variety of challenges in living shorelines science, which
are summarized in Table 2 and compared with practitioner-identified challenges. First,
there is a critical need for quantitative assessments of long-term structural integrity of
living shorelines, as well as research into their maintenance requirements and success in
adapting to rising sea levels. Another widely cited knowledge gap is a lack of projects
directly comparing the effects of “green” or natural versus “gray” or artificial structures.
The incorporation of any artificial element into a living shorelines design is controversial
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 37
Table 2. Commonly identified challenges and knowledge gaps in living shorelines research and litera-
ture, grouped by number of interviews in which each was discussed: (A) =10; (B) 5 to 9; and (C) < 5.
Mentions Mentions
Common needs & (Stakeholders (Literature
challenges Examples & Action needed n= 23) n= 50)
Long-Term Need for 10+ years of data on living 13 (57%) 31 (62%)
Monitoring shorelines durability, adaptation,
ecological response, & ecosystem
services
Demonstration Pilot projects in new communities and 13 (57%) 16 (32%)
Projects varied coastal contexts
Engineer Training Design guidelines, standardized methods, 13 (57%) 18 (36%)
and Resources and training/certification programs in
natural infrastructure
Communications Social media outreach, video projects, 12 (52%) 14 (28%)
Campaigns community environmental education
Green-to-Gray Need for direct comparisons of man-made 11 (48%) 25 (50%)
Spectrum and natural structures and case studies
in high-energy environments
Cross-Disciplinary Increased communication and 11 (48%) 29 (58%)
Networks data/results sharing between scientists
and professionals, policymakers
Success Rate Need for data on living shorelines’ efficacy 11 (48%) 25 (50%)
in achieving long-term goals (i.e.,
coastal protection, habitat value)
Visualizations Maps, imaging, drone photos, virtual 10 (43%) 3 (6%)
reality, models, apps; any way to display
projects and gain community attention
Consistent Disagreements on what habitat types, 9 (39%) 9 (18%)
Definitions coastal contexts, and structural
elements constitute living shorelines
West Coast Regional compilations of shoreline designs 9 (39%) 10 (20%)
Guidelines & appropriate for different settings,
Examples expanding on successes and failures
Permit Streamlining Pilot-scale, streamlined permitting with 8 (35%) 14 (28%)
easily accessible checklists to enable
more efficient preparation
Environmental Need for outreach in under-served 5 (22%) 10 (20%)
Justice & Equity communities
Dialogue Between Sharing concerns, stories, goals, successes, 5 (22%) 11 (22%)
Communities challenges
Assessment of Public = Stakeholder outreach (i.e., public 5 (22%) 22 (44%)
Perception meetings, webinars, surveys)
Documentation of Availability of information (material cost, 4 (17%) 20 (40%)
Timeline & Cost permitting timelines, project
maintenance) to streamline future work
Regulatory Agencies —_ Increased advocacy to demonstrate 3 (13%) 20 (40%)
importance of living shorelines and
maintain funding
Building Ground-up Increasing community support for healthy 2 (9%) 9 (18%)
Demand
natural coastlines and extending
support beyond scientific community
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38 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Calgary =
) Legend
Vancouver
Stakeholder Interview
Seattle
Number of features
Montreal
9 -:
Toronto
Detroit Pion
Chicago ba @
=
Newyork
Denver ladelphia
1
St Louis Washington
ae ¢
Francisco Literature Source
°
1a, Number of features
Lo ey Atlanta
Dallas ° 3 > 6
= *
Houston + * 3
*
Monterrey Miami
Havana
——___— a ___
Q 500 1000km
Guadalajara
Mexico City owt 5
Port-au-Prince =\ 4 of
Esri, Garmin, FAO, NOAA, USGS, EPA | Esri, Garmin, FAQ, NOAA, USGS, EPA \==yr=]
Fig. 1. Map depicting geographical distribution of interviewees and literature sources consulted in this
study. Stakeholders interviewed are in red; literature sources are in black.
among living shorelines practitioners (Boudreau et al. 2018; Moosavi 2017). While some
advocates of hybrid green-gray approaches are encouraged by the durability of artificial
elements in the face of high wave energy, many biologists are concerned that small-scale
habitat restoration adjacent to armoring will add little ecological value and do nothing
to address the problem of hardened shorelines.' Living shorelines themselves, when in the
proper coastal setting, have been documented to be more effective in reducing the impacts
of storms than armored structures (Bilkovic et al. 2016). However, in open coast, high
energy environments, success is limited (Walker et al. 2011). More research into these high-
energy coastal contexts, especially during extreme weather events, is needed (Gittman et al.
2015; Hanak and Moreno 2012; Saleh and Weinstein 2016). Detailed risk assessments and
hazard modeling are imperative in understanding the durability of living shorelines into
the future and assessing the role they will play in reducing flood risk (Aerts et al. 2018;
Reguero et al. 2018; Russell and Griggs 2012).
Long-term monitoring of ecosystem services has been highlighted by numerous sources
as another essential component to living shorelines success, especially regarding wave at-
tenuation, faunal community response, and carbon sequestration (Benayas et al. 2009;
Bilkovic et al. 2016; Davis et al. 2015; Gittman et al. 2016a; Patrick et al. 2016;
Simenstad et al. 2006; Zeigler et al. 2018, Ridlon et al. 2021). These long-term data
convey the success or failure of nature-based infrastructure at meeting its coastal re-
silience goals, and are needed to assure coastal property managers, landowners, and poli-
cymakers that their investment in natural alternatives to shoreline armoring is worthwhile
(Baptist et al. 2019; Sutton-Grier et al. 2018). Next steps include streamlining communica-
' Pilkey, O.H., Young, R., Longo, N., Coburn, A., 2012. Rethinking Living Shorelines. Program for
the study of developed shorelines. Program for the Study of Developed Shorelines, Western Carolina Uni-
versity, 10 pp. Available from: http://www.oyster-restoration.org/wp-content/uploads/2012/06/Pilkey-et-
al.-Final-LS-White-Paper.pdf. Accessed 29 January, 2022.
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 39
tion between science and management and improving public perception of living shorelines
(Bragg et al. 2021; Currin 2019; Smith et al. 2020; Waltham et al. 2021)*. Clearer com-
munication of living shoreline benefits to the public using specific examples will establish
ground-up demand and lead to the institutional capacity building required for larger-scale
implementation.’ In short, a number of logistical and sociological hurdles underlie the
major knowledge gaps in living shorelines research, indicating areas where support is most
needed.
Many of the major challenges and barriers to living shorelines advancement identified in
the literature review corresponded with the findings of the interviews (Table 2). Practitioner
responses to interview questions varied to some extent by discipline, but many responses
were common or interconnected among interviewees. Fig. | indicates the geographi-
cal range of interview participants; most practitioners were from northern or southern
California (Table 1), reflecting the clustered distribution of living shorelines projects ob-
served in the literature review. The majority of interviewees and research articles consulted
both indicated that one of the most pressing challenges is a lack of long-term data evalu-
ating how living shorelines projects are meeting their specific goals, particularly along the
Pacific coast given that living shorelines have historically been concentrated in East coast
systems. Living shorelines projects are often monitored for a few years past their imple-
mentation, generally up to about five years with few exceptions. There is a simultaneous
need for more demonstration projects to begin and for monitoring of existing projects to
continue, a need that has been consistently identified over the past decades and prompted
many suggestions from interviewees. West coast living shorelines studies require further re-
search into project lifespan, standardized timelines and monitoring methods, and effective
restoration materials in different coastal settings. A growing network of professionals and
a collaborative effort to share data and methods is needed to guide future research and
streamline restoration processes in the future. As is further described in the discussion, in-
terviewees identified several approaches to increase community investment in restoration
projects for long-term persistence and positive perceptions of new projects.
Interviewees across multiple disciplines and organization types identified a great need
for capacity building when it comes to living shorelines restoration, especially professional
training programs. Many practitioners mentioned a lack of engineers trained in nature-
based infrastructure and familiar with its unique benefits and challenges, a limitation also
identified various times within the literature review. For living shorelines to become a more
widely used engineering practice, long-term studies of durability, adaptation, and main-
tenance needs are also essential, especially in high-energy environments. Specific regional
design guidelines should be created to distinguish which techniques have succeeded and
failed in a given coastal setting. These specifications can inform training and information
sessions for interested practitioners and can refine the method selection process when de-
signing future projects.
Improved communication with regulatory agencies was also identified by multiple prac-
titioners and various literature sources as an area where further work is greatly needed.
: Belcher, A., Bezore, R., Covi, M., Yusuf, W., 2019. Living Shorelines: Barriers and Promo-
tion: Accomack County, VA. Old Dominion University. 10pp. Available from: https://digitalcommons.
odu.edu/cgi/viewcontent.cgi?article=1019&context=odurc-presentations. Accessed 29 January, 2022.
* Restore America’s Estuaries, 2015. Living Shorelines: From Barriers to Opportunities. Arlington,
Virginia. 55 pp. Available from: https://estuaries.org/wp-content/uploads/2019/02/Living-Shorelines-
From-Barriers-to-Opportunities.pdf. Accessed 29 January, 2022.
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40 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Policymakers rely on concrete evidence that living shorelines work as intended, and this is
not achievable without ample scientific support and effective communication. Maintain-
ing dialogue between science and management will improve regulatory agencies’ under-
standing of living shorelines projects as well as researchers’ understanding of permitting
requirements. Streamlining the permitting process for future projects will enable new living
shorelines projects to get off the ground more efficiently and will make them an acces-
sible strategy for more communities and small nonprofits. Finally, increased community
engagement and data sharing is needed to shift public perceptions of living shorelines and
promote the concept of natural beaches and their benefits.
To address the challenges listed above, interviewees proposed a wide range of solutions
from innovative restoration studies to educational programs and communications cam-
paigns. The most commonly mentioned approaches are outlined in Table 3, as well as the
prevalence of these approaches in living shorelines literature. The discussion and recom-
mendation section overviews many of the most commonly referenced suggestions for spe-
cific project types, but several one-off suggestions are listed in Table 3 as well. Some of these
include unconfined sediment placement from dredge material as a method of sea level rise
mitigation, analysis of nature-based infrastructure in groundwater studies, where adapt-
ability is a clear advantage over armoring, and research into natural irrigation techniques
for restoration projects during drought periods. Other suggestions were specific to oyster
restoration, including the development and use of natural reef balls, the implementation
of shell recycling programs and networks, and the creation of higher-relief oyster beds for
greater structural stability. Finally, increasing the accommodation space between coast-
lines and hardened structures to accommodate wave action was identified specifically in
one interview as a valuable research area, though this echoed the needs outlined by many
interviewees.
Discussion
Both the literature review and stakeholder interview components of this study provided
insight into current limitations in living shorelines science, as well as priority areas for fu-
ture research. Interviewees suggested a variety of strategies, projects, and programs needed
to address these challenges. They further identified areas where available funding can be ef-
fectively placed to create a scalable impact. These recommendations built upon many of the
conclusions outlined within living shorelines literature and centered on the five following
topics: supporting innovative demonstration projects, supporting engineer trainings and
needs, funding long-term monitoring, investing in communications and media projects,
and prioritizing community engagement. The focus areas identified through this analysis
provide a guide for future restoration and funding efforts.
Supporting Demonstration Projects with Innovative Methods.—Supporting pilot
projects in their early research and development stages was one of the most frequently
suggested ways to make a scalable impact in living shorelines research. Lack of fund-
ing for initial implementation of new adaptation strategies remains a major barrier, es-
pecially in Northern and Central California (Moser et al. 2018). By funding prelimi-
nary research in its academic and/or early planning stages, strategically placed funding
and community engagement can help demonstration projects get off the ground. With
effective communication and documentation throughout the project, this initial support
can then be used to leverage public funds on a larger scale (e.g., grants from state and
federal agencies) (Fitzsimons et al. 2020). Furthermore, to encourage restoration efforts on
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 41
Table 3. Opportunities for supporting innovative restoration techniques and programs identified by
interviewees and within literature, grouped by number of interviews in which each was discussed: (A) > 10;
(B) 5-9; (C) 2-5; (D) one-off suggestions within interviews.
Mentions Mentions
Methodologies & (Stakeholders (Literature
Research areas Examples n= 23) n= 50)
Green-gray hybrid Studies directly comparing benefits and 11 (48%) 25 (50%)
structures drawbacks of nature-based
infrastructure, and/or assessing the
incorporation of limited man-made
materials in high-energy environments
Open coast and Studies assessing offshore vegetation 10 (43%) 6 (12%)
offshore studies efforts (1.¢., kelp and eelgrass
restoration) and resulting changes in
sediment deposition, especially as
shorelines migrate
Hazard and risk Predictions for long-term adaptation and 7 (30%) 12 (24%)
modeling durability, addressing rising sea levels
and ability to withstand disturbance
High wave energy New approaches to restoration in 7 (30%) 21 (42%)
settings high-energy environments
Community science Involving the local community in 6 (26%) 9 (18%)
long-term data collection to both
educate and build a collaborative
dataset
Identifying future Projects that plan ahead and identify 6 (26%) 13 (26%)
habitat habitat/land areas that will be needed
for managed retreat
Cobble studies Recommended by engineers: research into 5 (22%) 5 (10%)
the use of cobble as a natural
infrastructure material (e.g., durability,
movement)
Preparation for Providing food, arranging webinars, 5 (22%) 3 (6%)
public meetings bringing people together
Dune revegetation Recommended by engineers: research into 4 (17%) 4 (8%)
revegetated dunes and how they grow,
accumulate sediment, and protect
shores over time
Ecosystem service Studies calculating the economic value of 3 (13%) 13 (26%)
quantification services provided by restoration (water
clarity, biodiversity, storm protection)
Extreme weather Studies addressing ability to protect 3 (13%) 16 (32%)
events against storm surges
Experimental design Studies with randomized, replicated 3 (13%) 10 (20%)
experimental designs
Shell recycling Building a growing network of local 2 (9%) 3 (6%)
programs restaurants and businesses as partners,
providing shell for oyster restoration
Multiple habitat Connecting as many habitats as possible 2 (9%) 9 (18%)
types from subtidal to upland
Dredge sediment One USACE program, “Innovative Shore 1 (4%) 6 (12%)
monitoring and
placement
Protection”, once monitored dredge
sediments and needs replacement.
These sediments could be put to use in
sea level rise mitigation efforts.
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42
SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Table 3. Continued
Mentions Mentions
Methodologies & (Stakeholders (Literature
Research areas Examples n= 23) n= 50)
Community-based Collaboration between local government 1 (4%) 2 (4%)
subsistence areas and community, i.e., indigenous fish
ponds and community subsistence areas
in HI
Natural reef balls Alternatives to artificial reef balls used in 1 (4%) 3 (6%)
oyster restoration
High-relief oyster Need for taller oyster bed structures given 1 (4%) 2 (4%)
beds the low relief of Olympia oyster beds;
Baycrete may be a helpful material
Groundwater-based Using nature-based infrastructure in 1 (4%) 3 (6%)
natural groundwater, where adaptability is
infrastructure important and armoring is undesirable
Accommodation Projects focused on increasing the amount 1 (4%) 0 (0%)
space of space between wave action and
hardened structures
Stormwater Using stormwater to maintain restored 1 (4%) 1 (2%)
irrigation habitats, potentially used in a
larger-scale project such as a horizontal
levee
Drought studies Natural irrigation techniques for drought 1 (4%) 2 (4%)
periods where restored vegetation may
begin to die off
privately owned lands, economic incentives should be promoted and publicized to attract
the attention of private landowners who may otherwise not have interest in, or knowledge
of, implementing a living shoreline on their property (Scyphers et al. 2020). The next few
paragraphs detail several research areas that should be a priority in coastal restoration
based on their innovative methodologies.
First, projects involving risk assessment and evaluating the potential for managed re-
treat are greatly needed. Many practitioners reiterated the challenge of a highly urbanized
and developed Pacific coastline, especially in southern California, which requires restora-
tion approaches that cannot be applied directly from case studies in other regions. Adding
habitat value to these hardened coastlines is crucial, as is planning for managed retreat in
the face of sea level rise. Interviewees consistently identified risk assessments as a way to
increase scalability of living shorelines. Sea level rise modeling and incorporating extreme
weather events will inform next steps, including site selection for future pilot projects. It will
also aid in identifying “future habitat” where coastal habitat restoration efforts should be
focused to transition to higher sea levels; this can range from efforts to convert agricultural
land that will be unusable in the future or removing infrastructure currently in place that
will not withstand sea level rise. These risk assessments will further aid in developing esti-
mates of cost and shoreline protection value, a key piece of information for policymakers
(SCC et al. 2010; Reguero et al. 2018).
Research assessing living shoreline success in high-energy environments is also needed
to inform management strategies, specifically restoration site selection, project design, and
expectations for structural persistence and maintenance. Living shorelines have generally
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 43
been limited to low-energy areas such as back bays in order to introduce nature-based
infrastructure in areas with greatest potential for success. The focus must now be ex-
tended to regions where wave energy and erosion are high, in order to further demon-
strate their efficacy to engineers, policymakers, and other stakeholders involved in coastal
management. In these high-energy areas, several methodologies must be considered and
directly compared. This is especially true in systems where “gray” infrastructure is more
commonly used, readily available, or easy to implement. Considering the use of hybrid
“green-gray” structures may be a valuable first step in increasing resilience within highly
urbanized communities, while simultaneously increasing visibility of and engagement with
living shorelines (Moosavi 2017; Silvertooth et al. 2019). For example, revegetation efforts
along a horizontal levee or habitat restoration adjacent to a seawall already in place will
increase ecosystem services and present a first step in using natural structures as an alter-
native or draw attention to removing the armoring itself. Similarly, using a breakwall in
conjunction with oyster restoration was found to enhance ecosystem recovery in a high-
energy environment in Florida (Safak et al. 2020). These approaches must be considered
very carefully, given that many researchers are well aware of the scant ecological value of
projects closer to the “gray” end of the spectrum and caution that the habitat restora-
tion is more of a disguise for armoring than an adaptive approach to coastal resilience
(Smith et al. 2020)!.
To better inform engineers, policymakers, and other stakeholders involved in coastal
management, studies directly assessing benefits and drawbacks of green versus gray struc-
tures in different Pacific coast systems would be highly useful. Beyond establishing pilot
projects and monitoring their success, a coordinated effort is needed across the Pacific
coast to test green infrastructure in a series of high-energy coastal environments. This type
of regional study would provide replicated data to engineers, geomorphologists, and pol-
icymakers that can inform which conditions are ideal for fully natural solutions such as
dunes and, on the other hand, which conditions may require an understory of rock. It
would furthermore help identify instances in which using durable hybrid elements as one
component of a living shoreline might offer a better, or transitional solution. For exam-
ple, Baycrete/ECOncrete (concrete mixed with natural materials such as shell or sand)
may present an opportunity to construct high-relief oyster beds, considering the limited
bed size of the native Olympia oyster. Projects that entail standardized monitoring metrics
over an extended period (10+ years) and clearly communicate their results to scientists
across the Pacific coast are a funding priority based on the findings of our interviews and
literature review.
In addition to advocating for studies in high-energy environments, many practition-
ers and particularly engineers expressed that research on the open Pacific coast provides
further opportunities for innovation. Most interviewees with this suggestion worked at
the interface of science and management at relatively large scales, indicating that these
projects are especially important next steps in the big picture of advancing coastal re-
silience. Some specific examples include restoration of offshore vegetation, including kelp
and eelgrass; studying their impacts on sedimentation patterns and wave attenuation will
become increasingly important with sea level rise and shoreward migration. Projects link-
ing increased numbers of habitat types from upland to offshore should also be given pri-
ority, as they will provide a higher degree of structural diversity, thereby increasing habitat
value and wave attenuation (Zeigler et al. 2018). These types of open coast studies are
also important in better understanding engineering needs in higher-energy environments
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44 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
(Walker et al. 2011).4 Compiling a coordinated set of data to inform the restoration tech-
nique selection under a given set of physical and biological conditions should be a priority
to fill this knowledge gap and provide resources for future practitioners.
Finally, demonstration projects with an experimental design, especially a randomized
replicated design, are needed to reinforce scientific support behind living shorelines projects
(Gellie et al. 2018). Replication along the Pacific coast will establish the settings in which
different approaches are most effective as well as their projected likelihood of success. Data
sharing among these projects and dialogue between the communities in which they take
place should be facilitated. The results of these studies can be combined to contribute to a
regional guide outlining different techniques and the best contexts in which to use them.
Supporting Engineer Trainings and Needs.—The low number of engineers working
on living shorelines projects was identified in the literature review as a common chal-
lenge for restoration efforts across the U.S. (identified in 36% of sources reviewed; see
Table 2). A need for more engineer involvement on Pacific coast projects in particular
was emphasized by a majority of practitioners interviewed (57%; Table 2). In the con-
text of the interviews, the definition of engineers included geomorphologists with exper-
tise in the structural dynamics of coastal ecosystems, geologists studying shoreline pro-
cesses, and GIS practitioners assessing structural changes over time. To increase engi-
neer interest and involvement in living shorelines projects, more certified training pro-
grams in natural infrastructure are needed as well as targeted recruitment efforts through
university interest groups, seminars, and collaboration with interdisciplinary professional
societies. One program that may present an opportunity for collaboration is the En-
gineering with Nature® (EWN) program developed by the U.S. Army Corps of Engi-
neers (USACE; Kurth et al. 2020). EWN has partnered with nonprofits such as The
Nature Conservancy and its Natural Infrastructure Initiative, providing a model upon
which recruitment and partnership efforts could be based. These partnerships also pro-
vide a unique opportunity for future collaboration on living shorelines projects; restora-
tion practitioners can benefit from engineers’ perspectives, and direct work with the US-
ACE can help to streamline permitting and foster connections between coastal managers
and engineering firms. Communications projects drawing attention to and building upon
these existing resources could go a long way in generating interest and expanding training
programs.
Two concerns raised in interviews with both engineers and policymakers were related to
structural persistence of non-hardened structures and the efficacy of small-scale projects
in achieving the desired restoration goals. Overall, these practitioners expressed a need
for research that can provide data showing that nature-based approaches are both practi-
cal and feasible. First, engineers identified a need for projects documenting the ability of
non-hardened structures to effectively persist over time and stabilize coasts, such as oys-
ter restoration projects as a substitute for seawalls, or dune restoration using driftwood
and cobble. Another area of interest among engineers interviewed was the influence of
grain size on dune stability and vegetation growth and faunal communities. Second, both
engineers and policymakers acknowledged that smaller-scale projects are considered valu-
* Miller, J.K., Rella, A., Williams, A., Sproule, E., 2015. Living Shorelines Engineering Guide-
lines. Stephens Institute of Technology, 102 pp. Available from: http://stewardshipcentrebc.ca/PDF_
docs/GS_LocGov/BkgrdResourcesReports/living-shorelines-engineering-guidelines.pdf. Accessed 29 Jan-
uary, 2022.
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 45
able within their field, but only so as they lead to larger-scale projects that increased the
area of restoration. This perception may overlook the importance of these pilot projects
for demonstrating efficacy and usefulness of nature-based infrastructure. Finally, a major
knowledge gap emphasized in both literature and stakeholder interviews was a lack of di-
rect green vs. gray comparisons. In Kurth et al. (2020), scientists recommended conducting
formal tradeoff analyses comparing shoreline armoring with natural infrastructure; this
was supported by several other sources indicating a need for longer-term data on stabil-
ity, cost, and maintenance over time for both hard and soft defenses from sea level rise
(Bragg et al. 2021; O’Shaughnessy et al. 2020; Sutton-Grier et al. 2018)°. Targeting these
questions and research gaps can answer important questions within the field of natural
resources engineering and establish guidelines for long-term design.
Continuing to Fund Long-Term Monitoring.—The most frequently identified need
among all practitioners interviewed (57%) and supported strongly by literature sources
(62%; Table 2) was increased funding for long-term monitoring especially for Pacific coast
projects. Datasets of 10+ years are integral to understanding living shorelines’ success from
cross-disciplinary perspectives (Bayraktarov et al. 2016; Waltham et al. 2021). The primary
goal of engineers is to create long-lasting structures, and insufficient data on persistence of
living shorelines is a barrier in recruiting engineers to work with nature-based infrastruc-
ture. From a biological standpoint, ecological communities fluctuate both seasonally and
annually, and long-term monitoring data is needed to distinguish restoration effects from
interannual variation. Maintenance requirements and costs over time are also important
information for property owners, local governments, and potential mitigation funders, and
cannot be properly assessed without long-term studies. Given this need for long-term data
and the research priorities identified in our study, priority should be given to projects as-
sessing the structural persistence of nature-based infrastructure, with a standardized set of
metrics such as elevation, habitat cover, ecological community responses, and maintenance
requirements. Potential funders should seek out restoration efforts with concrete commu-
nications goals or with coordinated projects in other geographic locations.
Small foundations are integral to funding monitoring programs, especially in partner-
ship with universities and local nonprofits. Having a long-term dataset was often refer-
enced by practitioners when discussing scalability, as it provides more concrete evidence
of the efficacy of living shorelines to planners and managers. Providing funding for unique
monitoring approaches (i.e., community science programs, volunteer data collection days,
video and drone monitoring) would be a creative strategy to increase visibility of living
shorelines projects and to incorporate community engagement. Because long-term moni-
toring requires continual efforts to secure funding, leveraging funds in this way is a strategic
approach that will contribute to larger-scale research and secure support for smaller orga-
nizations that may not have time or capacity to continually apply for short-term funding.
Investing in Communications & Media Projects.—Communications campaigns were
also recommended by a majority of interviewees (12 practitioners or 52%: Table 2). This
need was identified in the literature review as well, within 14 sources or 28% of the liter-
ature reviewed (Table 2). The strong relative emphasis on communications campaigns in
interviews highlights a need that may be underrepresented in published literature. While
: Myszewski, M., Alber, M., 2016. Living Shorelines in the Southeast: Research and Data Gaps. Athens,
Georgia. 203 pp. Available from: http://southatlanticalliance.org/wp-content/uploads/2016/09/Living-
Shorelines-in-the-Southeast.pdf. Accessed 29 January, 2022.
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46 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
interviewees indicated that communication is progressing among scientists studying differ-
ent habitats (dunes and oyster beds, for example) and between scientists and other profes-
sionals, improvement is still needed. Improved communication begins with better consen-
sus on the definition of living shorelines themselves. The interdisciplinary nature of living
shorelines work often means that different experts have different interpretations of which
projects can be considered living shorelines. Scientists need to continue building a network
to compile case studies regionally and to be aware of advances in disciplines not directly
related to their own. The ability to compare methodologies, materials, coastal context, and
successes or failures is needed for all types of living shorelines restoration (Baggett et al.
2015; Bayraktarov et al. 2016; Molino et al. 2020; Zeigler et al. 2021). Better communica-
tion is also especially needed in conveying the importance of living shorelines to the public
(Bragg et al. 2021; Molino et al. 2020).
The ability of coastal communities to visualize proposed changes to their local beaches
and properties is essential in gaining public support, whether it be through a projected im-
age presented at a local meeting, a video project or virtual reality experience, or an exam-
ple of a successful pilot project in a similar coastal context. Several practitioners suggested
unique approaches to providing visualization for proposed or ongoing projects. Social me-
dia outreach is an important first step, and regularly providing educational information
and project updates will shift public knowledge and perceptions. Other creative approaches
to outreach include showing drone imagery of restored sites, models of proposed projects,
or simulations of proposed changes on a phone app. These are all engaging examples of
showing local communities, policymakers, and landowners how their local coastline will
change and highlighting benefits of natural shorelines.
Communicating benefits to local management and communities is an important step
in gathering more ground-up demand for living shorelines projects, and communications
campaigns are particularly effective if economic incentives are highlighted. While the eco-
logical value and ecosystem services of living shoreline restoration are important, it is es-
sential to communicate the precise value of increased fish production, water quality im-
provements, wave attenuation, and other benefits. Projects translating ecosystem services
into economic values, with specific plans for communicating these findings, should be con-
sidered for funding as they will drive increased demand and leverage future funds.
Prioritizing Community Engagement.—Practitioners agreed that community engage-
ment is essential for living shorelines success and should continue to be a major focus in
future restoration efforts. Increasing connection with local stakeholders, providing an out-
let for them to express their concerns, keeping them informed about recent scientific find-
ings, and demonstrating the services provided by restored habitats are all necessary steps in
maintaining this connection. Community engagement has been identified to both increase
stewardship of existing and restored habitats as well as to increase understanding and sup-
port for restoration and associated funding (Bragg et al. 2021; Chang et al. 2019; Scyphers
et al. 2020)*. Efforts to promote volunteer-based or community science also provide an op-
portunity for long-term data collection and increase engagement with restoration projects;
such stakeholder engagement efforts have been shown to change the perceptions and views
of community members regarding restoration projects (Boudreau et al. 2018; Josephs and
Humphries 2018).
An increased focus on nature-based coastal resilience measures is especially needed in
communities affected disproportionately by sea level rise and environmental degradation.
In under-served communities, socioeconomic factors often limit access to environmental
education and to nature itself. Public perception and community well-being can both be
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LIVING SHORELINES STAKEHOLDER INTERVIEWS 47
supported through educational measures, such as field trips to restored beaches for stu-
dents or discussions with local fishermen about increased fish presence post-restoration.
Agencies such as the California State Coastal Conservancy (SCC) have been working in
recent years to re-center their mission on environmental justice and equity; these efforts
are needed in all regions to increase scalability of public interest and collaboration.® Build-
ing ground-up demand for living shorelines projects starts with building a connection to
projects within communities, then enhancing dialogue between communities.
Fostering connections between communities and government in joint projects can have
lasting positive impacts, especially when local knowledge enhances resource management.
This can be seen in the collaboration between indigenous communities and local gov-
ernment in community-based subsistence areas and biocultural resource management in
Hawaii (Chang et al. 2019; Winter et al. 2018). Indigenous fish ponds in Hawaii serve as
a valuable example of community-based adaptive management and cross-disciplinary col-
laboration. They highlight the importance of supporting indigenous group autonomy and
enhancing productive relationships between stakeholders within a community.
Lessons Learned for Future Efforts. —Both the literature review and interview portions
of this study reaffirmed a need for continued outreach and discussion with stakeholders in
living shorelines restoration, including local community members and volunteers, home-
owners, students, scientists, engineers, members of state agencies and nonprofit organi-
zations, and more. Engaging with stakeholders, including coastal landowners and other
community members, early in the restoration process allows for priorities and commu-
nity values to be established early on and persist throughout all project stages (Fitzsimons
et al. 2020; Josephs and Humphries 2018). Taking advantage of every opportunity possible
in bringing people together and enhancing dialogue between communities, from meetings
and webinars to accessible regional conferences, is essential (Bragg et al. 2021; Toft et al.
2017). Many interviewees were very enthusiastic about continuing stakeholder outreach
efforts, emphasizing the importance of diverse perspectives in discovering opportunities in
coastal communities. Increased representation is particularly needed moving forward for
native communities and communities of color. These groups should form a substantial part
of discussions on living shorelines, as their involvement is needed to address their concerns
and promote projects that advance their interests and well-being.
Our research and conversations also highlighted a need for increased communication
and data sharing among West coast living shorelines practitioners. Our interviews primar-
ily incorporated perspectives from the Pacific coast; many interviewees from this region
agreed that local restorations are heavily informed by work done in the Gulf and East
coasts, where living shorelines research, especially involving oyster restoration, is older.
Less is still known about restoring certain Pacific coast species such as the Olympia oys-
ter, or addressing challenges within unique coastal settings (1.e., open coast, higher en-
ergy environments). Supporting demonstration projects on the Pacific coast in new coastal
contexts, collecting long-term data, and sharing findings effectively among stakeholders
are all essential next steps in increasing the scale of living shorelines restoration in the
future.
° California State Coastal Conservancy (SCC), 2020. State Coastal Conservancy JEDI Guidelines in
Action. 5 pp. Available from: https://scc.ca.gov/files/2020/09/JEDI_Guidelines_In_Action_FINAL.pdf.
Accessed 29 January, 2022.
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48 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
Conclusions
After reviewing recent literature on living shorelines and gathering insight from a varied
group of cross-disciplinary practitioners, the following priorities were identified: (1) sup-
porting pilot or demonstration projects in their early research stages, (2) increasing avail-
ability of training programs for engineers utilizing nature-based infrastructure, (3) moni-
toring ecological and structural properties of living shorelines in the long-term (5+ years),
(4) communicating results, project significance, and visual resources to stakeholders within
and between communities, and (5) advancing the causes of environmental justice and
equity.
Acknowledgements
This work was supported by funding from the Honda Marine Science Foundation,
which has since been absorbed by the American Honda Foundation. We would like to
give special thanks to the Honda Marine Science Foundation board members Raminta
Jautokas, Jessalyn Ishigo, and advisors Sarah Sikich and Chris Yates for their substantial
input throughout all stages of this research. This would not have been possible without the
interest and support of all stakeholders interviewed in this study, and we thank them for
their time and insightful discussions.
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50 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
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Bull. Southern California Acad. Sci.
122(1), 2023, pp. 51-56
© Southern California Academy of Sciences, 2023
A New Maximum Length for the Bluebanded Ronquil, Rathbunella
hypoplecta (Zoarcoidei: Bathymasteridae)
Benjamin W. Frable,'* Jeremiah Dumford,* Alaina Conrad,” and John R. Hyde?
! Marine Vertebrate Collection, Scripps Institution of Oceanography, University of
California San Diego, La Jolla, CA, USA.
* California Department of Fish and Wildlife, San Diego, CA, USA.
3NOAA, National Marine Fisheries Service, Southwest Fisheries Science Center,
La Jolla, CA, USA
Abstract.—Throughout 2019, multiple large Rathbunella specimens were collected
from recreational hook and line fishermen off San Diego County, California, USA.
The individuals were larger than the previously reported maximum size for Rath-
bunella and exhibited a dark coloration known but not usually depicted for the genus.
Herein, we provide morphological and molecular evidence that these specimens are
Bluebanded Ronquils, Rathbunella hypoplecta, and we increase the maximum size of
the species to 242 mm standard length. We provide descriptions of the specimens and
the dark-phase coloration and demonstrate that dark and light-phase individuals are
not genetically different.
The bathymasterid genus Rathbunella Jordan and Evermann, 1896 is a relatively com-
mon demersal zoarcoid genus comprising two species found on the northeastern Pa-
cific coast from Marin, California to San Carlos, Baja California (Love and Passarelli
2020). Of the two species, Rathbunella alleni Gilbert, 1904 is distributed along the full
range, but is most common in central California from depths of 2-146 m, whereas R. hy-
poplecta (Gilbert, 1890) is known from Point Conception, California to Santo Tomas, Baja
California from depths of 9-136 m (Stevenson and Matarese 2005; Love and Passarelli
2020). The two species have been confused with some authors considering R. alleni as a
synonym of R. hypoplecta (Eschmeyer and Herald 1983); however, Stevenson and Matarese
(2005) redescribed the species and identified a suite of characters supporting its validity and
distinction from R. hypoplecta. Rathbunella alleni differs in having rougher scales, lacking
an accessory preopercular pore, having males with large canines and having a blue stripe
through the anal fin rather than blue bands and 1s generally considered to be rarer than R.
hypoplecta (Miller and Lea 1972). Rathbunella hypoplecta is the larger of the two species,
generally reported to reach a maximum size of 216 mm total length (TL) with R. alleni
around 161 mm standard length (SL) (Love and Passarelli 2020).
During 2019, California Department of Fish and Wildlife (CDF W) personnel collected
five Rathbunella specimens ranging from 248-277 mm TL (unpreserved) off San Diego
County, California, USA, that are larger than the previously reported maximum length for
the genus. Specimens were collected on hook and line by recreational anglers and recovered
by CDFW personnel sampling from fishing operations. Additional material was observed
but not retained from locations throughout San Diego County, CA. Historically, there has
been some confusion on the maximum size of R. hypoplecta. Barnhart (1936) reported a
* Corresponding author: bfrable@ucsd.edu
51
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52
Table 1. Morphometrics for newly collected Rathbunella hypoplecta specimens and those reported by
Stevenson and Matarese (2005). Cephalic measurements are reported as percentage head length, all others
are percentage standard length (SL). Means are in parentheses.
Unpreserved Total Length
Preserved Total Length
Preserved SL
Head length
Snout length
Upper jaw length
Eye diameter
Interorbital width
Body depth at dorsal-fin origin
Body depth at anus
Caudal peduncle depth
Dorsal-fin length
Anal-fin length
Pectoral-fin length
Pelvic-fin length
Predorsal length
Preanal length
Snout to anus
Snout to pectoral-fin base
Snout to pelvic-fin base
SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
New specimens
n=5
248-277
245-276
209-242
20.5—22.7 (21.5)
24.4-27.6 (26.3)
36.1—-40.7 (38.8)
18.6—20.7 (19.8)
14.0-17.1 (15.9)
14.4-15.6 (14.8)
15.2-17.6 (16.2)
6.9-7.4 (7.1)
76.2—79.9 (79.0)
54.1—-57.5 (55.9)
16.3-17.9 (16.8)
10.5—12.1 (11.1)
16.4-19.0 (17.3)
40.5—41.9 (41.0)
39.3-40.6 (39.8)
20.7—22.6 (21.9)
18.9-21.1 (19.7)
Steven and Matarese (2005) specimens
n=l1
116-175
20.4-23.7 (21.6)
16.5-27.0 (22.5)
29.6-38.6 (33.3)
18.9-24.9 (22.5)
10.8-15.4 (13.3)
13.3-15.2 (14.3)
15.0-17.4 (16.0)
7.2-8.6 (7.8)
76.8-80.8 (78.5)
51.3-57.9 (55.0)
15.7-18.8 (17.2)
10.2-13.1 (11.6)
18.0-20.3 (18.9)
40.3-45.5 (42.2)
38.3-42.8 (40.3)
21.1-25.1 (22.6)
14.9-23.0 (19.5)
maximum length to 8 inches (203 mm). Miller and Lea (1972) report a maximum total
length (TL) for the genus Rathbunella as “around 8.5 in.” or 216 mm. Fitch and Lavenberg
(1975) report the same maximum length of 216 mm, but state this may be a different species
as the largest they personally observed was 159 mm. Eschmeyer and Herald (1983) report
a smaller maximum size of 160 mm, likely from Fitch and Lavenberg (1975). Love (2011)
reports 161 mm as the maximum size and Kells et al. (2016) report 8.5 in., likely following
Miller and Lea (1972).
Stevenson and Matarese (2005) examined UCLA W60-64, 215 mm standard length (SL),
which is potentially the source for the previous reported largest specimen; however, this is
not recorded as the largest specimen in their study (Stevenson and Matarese 2005: Table 1).
This specimen is now LACM 51837-1 and is remeasured as 213 mm SL and 245 mm TL (W.
Ludt, pers. comm. 2019). Additionally, Stevenson and Matarese (2005) report the locality
in error as “100 mi off Long Beach”, as the verbatim locality in the UCLA catalog is “Los
Angeles Co.: off Long Beach, Horseshoe Kelp Bed”.
Based on morphological comparison, we determined that these large specimens repre-
sent R. hypoplecta and herein provide morphological and molecular support for our identi-
fication. We increase the maximum size for R. hypoplecta and the genus Rathbunella to 277
mm TL (unpreserved; Fig. 1A) and report this in Love and Passarelli (2020). Additionally,
four of the five specimens collected exhibited dark coloration likely matching the “Deep-
water Ronquil” a potential undescribed species mentioned by Eschmeyer and Herald
(1983) with a darker overall body color and bright blue bars between pairs of anal-fin rays,
while one specimen had the more common light coloration with a sandy body color with a
bright blue stripe through the anal fin. Stevenson and Matarese (2005) suggested that the
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NEW MAXIMUM LENGTH FOR RATHBUNELLA 53
Fig. 1. Large specimens of Rathbunella hypoplecta collected in San Diego County, California, USA. A)
SIO 19-56, 242 mm SL, largest individual, preserved; B) SIO 19-7, 217 mm SL, dark-phase coloration prior
to preservation; C) SIO 19-57, 209 mm SL, light-phase coloration prior to preservation. Scale bars equal
20 mm.
“deepwater” specimens do not represent a separate species but a regular color phase for R.
hypoplecta. We provide genetic comparisons of the dark and light color phase specimens
and conclude these are indeed different color phases of the same species.
Materials and Methods
Methods of counting and measuring follow Hubbs and Lagler (1958) and Stevenson
and Matarese (2005). Measurements were taken with digital calipers and are reported to
the nearest 0.1 mm. Fin element and vertebral counts were taken from digital radiographs.
Gill raker counts are presented as upper (epibranchial) + lower (ceratobranchial) rakers
on the anterior face of the first arch; the angle raker is included in the second count. Re-
cently collected specimens are compared with counts and measurements from Stevenson
and Matarese (2005). Where counts were recorded bilaterally, both counts are given and
separated from each other by a slash; the first count presented is the left count. Morpho-
metric values for the specimens are presented in Table 1. Specimens examined and reported
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54 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
are deposited in the Scripps Institution of Oceanography (SIO); institutional abbreviation
follows Sabaj (2019).
To further verify species identity, mitochondrial cytochrome c oxidase subunit I (COT)
sequences from the recent specimens were compared with publicly available sequences ob-
tained from Genbank. PCR reactions were prepared to amplify an approximately 655 bp
fragment of the cytochrome oxidase I (COI) mtDNA gene using an M13-tailed primer
cocktail COJ-3 from Ivanova et al. (2007). Reactions were prepared with a final concen-
tration of 2 mM each dNTP, 0.25 uM of each primer, 0.5 mg/ml Bovine Serum Albu-
min (BSA), and 0.5U of standard DNA polymerase. Following a denaturation at 94°C for
two minutes, 35 cycles of the following cycling were performed: 94°C for 30 sec, 55°C for
30 sec, and 72°C for 1 min. A final extension at 72°C for three min terminated the cycling.
Di-deoxy Sanger sequencing was performed in both directions with universal M13 primers
using BigDye Terminator v3.1 chemistry following manufacturer’s protocol. Sequences
were generated on an ABI3730 at the Southwest Fisheries Science Center. Sequences were
aligned and edited in Sequencher v4.8, and the resulting contig was compared to sequences
in GenBank using the BLAST function.
Results
Rathbunella hypoplecta (Gilbert, 1890)
Fig 1A—C, Table 1
Material.—Rathbunella hypoplecta, five specimens, 208.5—-242 mm SL, San Diego
County, California, USA: SIO 19-7 (Fig. 1B), 217 mm SL, 256 mm TL, Box Canyon,
33°19’ 6’N, 117°31/ 48” W, 78.6 m, hook and line, recreational fisherman, 25 March 2019;
SIO 19-55, 236 mm SL, 271 mm TL, Devil’s Reef, ca. 3.7 km (2.3 mi) off Leucadia, 33°03’
54” N, 117°31' 48” W, 85.4 m, hook and line, recreational fisherman, 5 May 2019; SIO 19-
56 (Fig. 1A), 242 mm SL, 276 mm TL, Box Canyon, 33°19’ 6”N, 117°03’ 54” W, 72.1 m,
hook and line, recreational fisherman, 9 May 2019; SIO 19-57 (Fig. 1C), 209 mm SL, 245
mm TL, Box Canyon, Devil’s Reef, ca. 3.7 km (2.3 mi) off Leucadia, 33°03’ 54”N, 117°20'
30” W, 87.1 m, hook and line, recreational fisherman, 18 June 2019; SIO 19-68, 225 mm SL,
261 mm TL, 3.2 km (2 mi) off Del Mar Fairgrounds, 32°58’ 36”N, 117°18’ 48” W, 69.5 m,
hook and line, recreational fisherman, 4 October 2019.
Diagnosis.—A species of the genus Rathbunella differentiated from its congener, R. al-
leni, by the following combination of characters: ctenoid scales weak with short cteni (vs.
long); preopercular pores 7 + 1 (vs. 7); first dorsal-fin pterygiophore inserted between first
and second neural spine; anal-fin membrane solid bright blue, sometimes in bands (vs. a
single stripe).
Description. —D V-—VI, 40-41 (45-46 total elements); A II, 32 (34 total elements);
P 15-17/16-17; Vertebrae 14 + 36 (one with 34), total 50 (one with 48); upper procurrent
caudal-fin rays 7; upper principal caudal-fin rays 7 (one with 6); lower principal caudal-fin
rays 6; lower procurrent caudal-fin rays 6 (one with 7); pored lateral-line scales 83-85. Head
rounded, cheek with scales; snout rounded, blunt, 24.4-27.6% HL; eye diameter 18.6—
20.7% HL; interorbital width 14.0-17.1% HL; jaws subequal, lips thick; mouth slightly
oblique and short, not extending past midorbit, upper jaw length 36.1-40.7% HL; teeth
in upper and lower jaw small, conical, some slightly recurved, a few larger teeth, no ca-
nines; small teeth on vomer and palatine; gill rakers stout with tooth patches, 3-4 + 10-11;
branchiostegal membranes joined broadly and free from isthmus. Preopercular lateralis
canal with 7 pores plus a single pore from secondary caniculus off anteroventral end of
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NEW MAXIMUM LENGTH FOR RATHBUNELLA ae
main canal; mandibular canal with 4 pores. Scales weakly ctenoid, over entire body except
just anterior dorsal-fin, head anterior of cheek and on fin membranes except dorsal and
caudal-fin bases. Lateral line originating posterior to opercular margin and terminating
before dorsal-fin insertion.
Coloration when fresh.—Four of the five retained specimens displayed a dark color
phase (Fig. 1A—B), likely the “Deepwater Ronquil” of Eschmeyer and Herald (1983),
whereas one (SIO 19-57; Fig. 1C) displayed the more typical light sandy color phase re-
ported in Rathbunella hypoplecta (Love 2011). The dark-phase specimens are generally
dark blue-black with indistinct lighter barring along the body. When fresh, anal fin with
iridescent royal blue bars nearly touching to give solid blue appearance to fin, fading to
dark gray-black in death (Fig 1B), no red bars evident between anal-fin membranes as de-
scribed in Eschmeyer and Herald (1983), tips of anal-fin rays black. The light color phase
sandy beige to grey ground color with more distinct darker barring along the body and
base of dorsal fin; dorsal fin dark grey-blue; when fresh, anal fin with bright royal blue
stripe down length covering distal two-thirds of fin fading to dark grey-black in death, tips
of anal-fin rays black (Fig. 1C).
Coloration in alcohol (Fig. 1A ).—Dark and light-phase specimens similar in preser-
vation. Head dark brown dorsally, cheek reddish-brown; body tan in ground with dark
brown chain extending along midbody to caudal peduncle; 10—12 indistinct dark brown
blotches on dorsal surface, extending into dorsal-fin element bases, blotches 2—3 elements
long. Dorsal fin dark gray becoming brown distally; anal fin light gray, rays light brown,
distal tips of anal-fin rays distinctly black; pectoral fin light gray becoming dark distally;
pelvic fins blue-black; caudal fin brown with thin dark margin.
Habitat.—These specimens were collected by recreational fishermen bottom-fishing
for rockfishes (Sebastes) on deep rocky reef habitats 3.2-4.8 km (2-3 mi) offshore in
69.5—-87.1 m of water.
Discussion
We resolved 649-652 bp of the COI gene for two specimens: SIO 19-7 and SIO 19-57
(GenBank Accession numbers: OP480876 and OP480877, respectively). BLAST results re-
cover 99.85—100.00% and 99.69-99.85%, respectively, matches of these sequences to vouch-
ers identified as Rathbunella hypoplecta on Genbank: EU400166 and GU440493, which
correspond to voucher SIO 03-60, and KF930350, corresponding to tissue KUT 494 and
voucher KU 28137. The next closest sequence is 88.89-88.99% similar (FJ165465) and
identified as Xiphister mucosus in Stichaeidae. Interestingly, this is more similar than se-
quences from other bathymasterid genera. Unfortunately, no public COI sequence exists
for R. alleni for intrageneric comparison. We also found that the COI sequences were
99.85% identical between the light-phase (SIO 19-57; Fig. 1C) and dark-phase specimens
(SIO 19-7; Fig. 1B) providing additional evidence that the dark-phase specimens do not
represent a distinct species.
Morphometrics and meristics are reported in Table 1. All five specimens fall within the
meristic and morphometric ranges reported for R. hypoplecta by Stevenson and Matarese
(2005) except some morphometrics just outside the range (Table 1). In the new specimens,
we found a slightly longer snout length (27.6 vs. 27.0% HL) and upper jaw length (40.7
vs. 38.6% HL), a slightly smaller eye diameter (18.6 vs. 18.9% HL), a wider interorbital
width (17.1 vs. 15.4% HL). In body measurements, we identified a slightly deeper body
at dorsal-fin origin (15.6 vs. 15.2% SL), a slightly shorter dorsal-fin base (76.2 vs. 76.8%
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56 SOUTHERN CALIFORNIA ACADEMY OF SCIENCES
SL), a slightly narrower caudal peduncle depth (6.9 vs. 7.2% SL) and a shorter snout to
pectoral-fin base (20.7 vs. 21.1% SL). The new specimens differ from R. a/leni in having
weakly ctenoid body scales (vs. strong), an accessory preopercular pore and the first dorsal-
fin pterygiophore inserted between the first and second neural spine (vs. anterior or dorsal
to the first neural spine).
Conclusions
This new material increases the known maximum size for the genus Rathbunella from
216 mm SL to 242 mm SL (276 mm TL) in preservation and 277 mm TL unpreserved.
Additionally, we used genetic comparisons of this material to determine that the potentially
undescribed “Deepwater Ronquil” of Eschmeyer and Herald (1983) is in fact just the dark-
phase coloration of Rathbunella hypoplecta.
Acknowledgements
We would like to thank the crew, recreational fishermen and CDFW staff for recovering
the specimens, W. Ludt (LACM) for measuring and photographing the LACM specimen
and L.E. Martin and M. Craig (NOAA) for assistance with sequencing. Further, we thank
M. Love (UCSB), D. Stevenson (NOAA) and R.N. Lea for assistance with initial identifi-
cation and discussion of the specimens.
Literature Cited
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Hubbs, C.L., and K.F. Lagler. 1958. Fishes of the Great Lakes region. Cranbrook Institute of Science
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Sabaj, M. H. (2019). Standard symbolic codes for institutional resource collections in herpetology and
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